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The aim of the present study was to investigate whether the phosphorylation of ERK1/2 in the rat lumbar dorsal horn and in the parvocellularis part of the paraventricular nucleus can be used to visualize neuronal activity. pERK1/2 fluorescence-immunohistochemistry is specifically suited to mirror neuronal activity in the pain pathway following an acute noxious stimulation. The rat hind paw was either stimulated by noxious heat or by a sequence of mustard oil and noxious heat. Two and 10 min after the thermal stimulation a 3–4-fold increase in cells with pERK1/2 immunoreactivity was observed in lamina I/II of the L3-L5 dorsal horn. The combination of mustard oil with heat led to a 5–6-fold increase in the pERK1/2 signal. The pERK1/2 immunoreactivity in the parvocellularis part of the paraventricular nucleus increased by 2-fold following the heat stimulus, with no further increase following the sequential mustard oil and heat stimulus. A pretreatment with the opioid analgesic morphine or the NMDA antagonist MK-801 markedly attenuated ERK1/2 phosphorylation in both areas of the pain pathway. The present findings support the concept that the pERK1/2 immunofluorescence signal can be used as a quantitative marker for sensitization or inhibition in the pain pathway at spinal and hypothalamic level. [ABSTRACT FROM AUTHOR]