The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process. Heat stimuli of above about 43 °C elicit a sensation of pain in humans, and initiate action potentials in nociceptive nerve terminals in humans and animals (Belmonte & Giraldez, 1981; Robinson et al. 1983). In experiments on isolated nociceptive neurones, heat was found to activate an inward current that has the properties expected from psychophysical and whole-animal experiments for the detector of painful levels of heat (Cesare & McNaughton, 1996; Reichling & Levine, 1997; Kirschstein et al. 1997; Nagy & Rang, 1999). Inflammation causes the threshold for the initiation both of a sensation of pain and of action potentials in nociceptive nerve fibres to fall to a lower temperature, a process known as sensitization (Treede et al. 1992). The heat-gated current also shows sensitization, in that the temperature threshold for activation of the current is lowered on exposure of the nociceptor to bradykinin, a nonapeptide released during inflammation (Cesare & McNaughton, 1996). Bradykinin sensitizes the heat-gated current by activating the ɛ isoform of protein kinase C (PKCɛ) (Cesare et al. 1999a). An important molecular sensor for heat is the receptor for capsaicin, which was recently cloned and named vanilloid receptor 1, or VR1 (Caterina et al. 1997). Capsaicin, the active ingredient of chilli peppers, excites nociceptive neurones (Baumann et al. 1991; LaMotte et al. 1992) resulting in a burning pain sensation (Buck & Burks, 1986). VR1 is a non-selective cation channel that is activated by a broad spectrum of stimuli, including capsaicin, heat, low pH and the endocannabinoid anandamide (Caterina et al. 1997; Tominaga et al. 1998; Zygmunt et al. 1999; Smart et al. 2000). Targeted disruption of the VR1 gene in mice produces animals that do not respond adversely to even high doses of capsaicin (Caterina et al. 2000), showing that VR1 is the only receptor for capsaicin linked to pain perception. VR1 knockout animals still respond to noxious heat, showing that other heat-sensitive nociceptor mechanisms exist in addition to VR1. A major difference between wild-type and VR1 knockout mice, however, was that the heat hyperalgesia observed in inflamed tissue was greatly reduced in the knockout, suggesting that VR1 is the principal mechanism responsible for heat hyperalgesia (Davis et al. 2000; Caterina et al. 2000). The experiments in the present study explore the role of PKC in the sensitization of VR1, using both nociceptive neurones and heterologously expressed VR1. In nociceptive neurones we found that the response to capsaicin is sensitized by PKC activation, in a similar manner to the response to heat, adding to the evidence that the same molecular mechanism is responsible for the response to the two stimuli. In HEK293 cells expressing VR1 (hVR1-HEK293 cells) the responses to heat, capsaicin, low pH and anandamide were found to be enhanced by PKC activation. The enhancement of current in response to capsaicin, in both nociceptive neurones and hVR1-HEK293 cells, was abolished in the absence of external Ca2+, consistent with the demonstration by other authors that desensitization of VR1 occurs by a calcium-sensitive process (Cholewinski et al. 1993; Docherty et al. 1996; Liu & Simon, 1996; Koplas et al. 1997). Finally, while heat, capsaicin, low pH and anandamide activate current through VR1, activation of PKC potentiates the effect of these stimuli but does not itself activate current, showing that PKC activation does not directly gate the VR1 channel. Preliminary reports of the data reported here have appeared elsewhere (Vellani et al. 1999).