The possibility that the class III antiarrhythmic drugs clofilium and d-sotalol might affect delayed rectifier potassium channels at the level of their gating currents was assessed with the whole-cell patch-clamp technique in guinea-pig isolated ventricular heart cells. Clofilium (up to 20 μM) and d-sotalol (1 μM) did not decrease the Na current, the L-type Ca current or the background K current iKl, but significantly depressed the time-dependent delayed outward K current iK. Clofilium partially decreased in a dose-dependent manner (1–20 μM) QON of intramembrane charge movements (ICM) elicited by a depolarizing pulse applied from a holding potential of −110 mV or following a 100 ms inactivating prepulse to −50 mV. D-sotalol (1 μM) also decreased QON. Channel density estimated from the clofilium-sensitive ICM closely matched that of the delayed rectifier channels. Clofilium and d-sotalol decreased QOFF seen on repolarization in a dose- and voltage-dependent manner. The kinetics of the decay of the OFF gating currents were not affected, and only the fast phase was depressed. In control conditions, QON availability with voltage was most of the time well described by two inactivating components. In the presence of clofilium and d-sotalol, a complex behaviour of QON availability was observed, unmasking additional components. The reactivation kinetics of QON after a 500 ms inactivating pulse to 0 mV was not affected. We conclude that delayed rectifier K channels significantly contribute to QON and QOFF of ICM in guinea-pig ventricular heart cells, besides Na and Ca channels, and that clofilium and d-sotalol directly interact with these K channels proteins by affecting their gating properties. Keywords: Intramembrane charge movements, K+ channels, native cardiac cells, class III antiarrhythmic drugs, clofilium, d-sotalol Introduction Intramembrane charge movements (ICM) in excitable cells result from molecular rearrangements of ion channel proteins under the impetus changes in the membrane potential. In mammalian cardiac cells, only sodium and calcium channels have been thought to give rise to ICM recorded with short depolarizing pulses, because of the much slower activation kinetics of the delayed rectifier potassium current (see Field et al., 1988; Bean & Rios, 1989; Hadley & Lederer, 1989, 1991a; Hanck et al., 1990; Shirokov et al., 1992; 1993). However, we have found more than two components of ICM in guinea-pig ventricular myocytes (Malecot & Argibay, 1996) which could not be entirely suppressed by the Na and Ca channels blockers lidocaine, tetracaine, nifedipine and D600 (Malecot et al., 1997a,1997b), although the underlying currents are blocked. However, compounds such as D600 or nitrendipine have been shown to be able to suppress all of the gating currents in heart cells, but at high concentrations that also affect potassium currents (e.g., Hadley & Lederer, 1991a). These results suggest that other channels might also contribute to ICM in heart cells. Moreover, recent studies of different expressed cloned potassium channels have revealed their fast gating currents (Larsson et al., 1996; Fedida, 1997), which are very similar in shape to those recorded in isolated heart cells. Therefore, we have re-investigated the pharmacological properties of ICM in freshly isolated guinea-pig heart cells and, in particular, we have tested the hypothesis of the presence of a component of ICM originating from the voltage-activated delayed rectifier K channels. Because several antiarrhythmic drugs have been shown to affect gating currents in many preparations, we have tested in this paper the possibility that the class III antiarrythmic drugs, clofilium and d-sotalol, blockers of iKs and of iKr (for review see Baro & Escande, 1993; Snyders, 1995), might affect delayed rectifier potassium channels at the level of their gating currents. We show here that ICM activating at positive potentials are sensitive to clofilium and d-sotalol at concentrations not inhibiting the Na or the L-type Ca channels but depressing the delayed outward K current in our recording conditions. To our knowledge, this is the first time that K channels are shown to significantly contribute to ICM in freshly isolated heart cells, besides Na and Ca channels. This new finding opens the door to a better understanding of the relationships between ICM and membrane excitability and to the future development of more powerful pharmacological compounds. A preliminary note on these experiments has been published (Malecot & Argibay, 1998).