Inhibition of cell metabolism is associated with significant changes in action potential duration. The aim of this study was to investigate the time course of the changes in action potential duration during metabolic inhibition and to determine what changes in membrane currents are responsible. The amphotericin perforated patch clamp technique was used to study membrane currents and voltage in single rabbit and human ventricular myocytes. In all myocytes inhibition of cell metabolism, induced by hypoxia (PO2 < 5 mmHg) or by addition of 100 microM 2,4-dinitrophenol (DNP), resulted in action potential shortening, which was accompanied by an increase in outward current, likely to be carried by ATP-regulated potassium channels. In about 65% of the rabbit and 50% of the human ventricular myocytes, however, action potential shortening was preceded by an initial prolongation. During this action potential prolongation, the L-type calcium current and the steady-state outward current remained unchanged. The transient outward current (Ito), however, was almost completely inhibited, suggesting that the action potential prolongation is caused by a decreased Ito. This interpretation was further supported by the observations that: (1) Action potential prolongation was found in all subepicardial myocytes, as was Ito, but only in a minority of the subendocardial myocytes. (2) Addition of DNP failed to cause action potential prolongation in subepicardial myocytes in the presence of 4-aminopyridine, a blocker of Ito. In conclusion, these data suggest that the phenomenon of action potential prolongation preceding action potential shortening during metabolic inhibition is mainly restricted to myocytes from subepicardial origin, and is due to a decrease in Ito.