In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na(+)-Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na(+)-Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia-reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mM yielded the following resting membrane potentials: -27.6+/-1.63 mV, -102.2+/-1.89, -86.5+/-1.03, -80.1+/-1.25, -73.6+/-1.02 and -66.4+/-1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia-reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura-2. These results demonstrated that after chemically induced hypoxia-reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mM [K+]o significantly reduced [Ca2+]i (7.5+/-0.32 vs. 16.9+/-0.55%); while depolarization (with either 0.5 or 15 mM [K+]o) significantly increased [Ca2+]i (31.8+/-3.21 and 20.8+/-0.36 vs. 16.9+/-0.55%, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na(+)-Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na(+)-Ca2+ exchanger inhibitor KB-R7943. During reoxygenation KB-R7943 (5 microM) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mM [K+]o: 2.2+/-0.40 vs. 10.8+/-0.14%) and in depolarized myocytes (in 15 mM [K+]o: -2.1+/-0.51 vs. 11.3+/-0.05%). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia-reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na(+)-Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.