To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca(2+) current (I(Ca,L)) by voltage and Ca(2+)-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses. During rectangular pulses, peak I(Ca,L) was significantly greater in EPI than in other cells. The inactivation of I(Ca,L) by Ca(2+)-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca(2+) sensitive, with Ca(2+) inactivation shifting the activation variables negative by approximately 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I(Ca,L) was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca(2+) influx.