Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0·01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 ± 8 ms in subendocardial (n = 30 myocytes), 251 ± 7 ms in midmyocardial (n = 30) and 204 ± 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1·59 ± 0·16 pA pF-1, n = 16) than in either subendocardial (1·16 ± 0·12 pA pF-1, n = 17) or midmyocardial (1·13 ± 0·11 pA pF-1, n = 21) myocytes (P < 0·05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0·50 ± 0·04 pA pF-1, n = 11) than in subendocardial (0·25 ± 0·05 pA pF-1, n = 9) or in midmyocardial myocytes (0·38 ± 0·05 pA pF-1, n = 11, P < 0·05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0·1 µM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35·7 ± 7·1 %, n = 11) than in midmyocardial (15·7 ± 3·8 %, n = 10) and subepicardial (20·2 ± 4·3 %, n = 11) myocytes (P < 0·05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.