Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+current (IK1) and delayed rectifier K+current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1and/or IKare responsible for the differences in action potential configuration between the two cell types. IK1in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between −80 and −30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N-shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1I-V relations. The characteristics of IKalso differed between endocardial and epicardial cells. The time course of growth of tail current of IK(IK,tall) (activation of IK) was significantly enhanced and that of IK,talldeactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IKactivation at +20 mV was 3,950±787 msec in endocardial cells and 2,746±689 msec in epicardial cells (p<0.05); the corresponding values for IKdeactivation at −50 mV were 1,041±387 msec and 1,959±551 msec, respectively (p<0.01). The voltage dependence of steady-state activation of IK,tallwas similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK(IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to −20 mV, IK,fullamplitude was 452±113 pA in endocardial cells and 578±135 pA in epicardial cells (p<0.05), and the corresponding values for IK,fulldensity were 2.86±0.73 and 4.21±0.83 μA/cm2, respectively (p<0.05). A nonstationary fluctuation analysis revealed that the amplitude of IKunitary current was similar between endocardial and epicardial cells (0.23±0.07 versus 0.22±0.03 pA,p=NS). Thus, the difference in whole-cell current amplitude of IKmight be due to the difference in the number of functional channels per myocyte. These data lead us to suggest that the differential characteristics of IK1and IKcontribute to the difference of action potential configuration between endocardial and epicardial cells.