1. Whole-cell and amphotericin-perforated patch-clamp techniques have been used to study the effects of hydrogen peroxide (H2O2) on action potentials and underlying ionic currents in single myocytes from the ventricles of adult rat hearts. 2. The results obtained differed markedly depending on the recording method utilized. Conventional whole-cell recordings, in which the myoplasm is dialysed with the contents of the pipette, failed to show any significant effects of H2O2 on the action potential or cell shortening. In contrast, when action potentials were recorded with the amphotericin-perforated patch method, H2O2 (50-200 microM) produced a marked prolongation of the action potential and an increase in cell shortening. 3. Voltage-clamp recordings with the amphotericin-perforated patch method showed that H2O2 caused no significant changes in either the Ca(2+)-independent transient outward K+ current (Ito) or the inwardly rectifying K+ current (IK1). 4. Application of tetrodotoxin (TTX; 8 x 10(-6) M), a Na+ channel blocker, largely inhibited the effects of H2O2 on the action potential. Moreover, anthopleurin A (4 x 10 (-7) M), which augments Na+ current (INa) by slowing its inactivation, mimicked the effects of H2O2 on the action potential of ventricular myocytes. These effects on INa were also blocked almost completely by TTX. 5. The hypothesis that H2O2 can augment INa by slowing its kinetics of inactivation was tested directly using ensemble recordings from cell-attached macropatches. These results demonstrated a significant enhancement of late opening events when H2O2 (200 microM) was included in the recording pipette. A corresponding slowing of inactivation of the ensemble INa was observed. 6. The possibility that protein kinase C (PKC) is an intracellular second messenger for the observed effects of H2O2 was examined using the blocker bisindolylmaelimide (BIS; 10(-7) M). Bath application of BIS prior to H2O2 exposure significantly delayed and also attenuated the development of the action potential prolongation. 7. These results demonstrate marked electrophysiological effects of H2O2 in rat ventricle. The dependence of these effects on recording methods suggests involvement of an intracellular second messenger, and the results with the PKC inhibitor, BIS, support this possibility. The most prominent effect of H2O2 on the ionic currents which underlie the action potential is a slowing of inactivation of the TTX-sensitive INa. Recent molecular studies have demonstrated a PKC phosphorylation site on the rat cardiac Na+ channel isoform and have also shown that PKC activation can slow inactivation of INa.