Experiments were performed to investigate the heterogeneity of the action potential and ion currents in left ventricular myocytes of the rabbit. Myocytes were isolated by enzymatic method. The sub-endocardial (Endo) and sub-epicardium (Epi) tissues were separated from the other region (midmyocardium, M) with a razor. Single cells in each region were obtained by gentle shaking and dispersing in a chamber filled with normal Tyrode's solution. The results showed that the action potential and the ion currents in the three layers were significantly different. M cells had a more pronounced spike-and-dome configuration, with a significantly larger phase 1 magnitude and plateau voltage. Action potential duration (APD) in M cells was longer than that in Epi or Endo cells. I(Ca, L) and I(to) in M cells were higher than those of Epi and Endo. On the contrary, I(K,s) in M cells was the minimum compared with those in the three LV walls. The differences in ion currents may well explain the heterogeneity of action potentials in M layers of the rabbit heart.