In single guinea-pig ventricular myocytes, we examined the stoichiometry of Na+-Ca2+ exchange (NCX) by measuring the reversal potential (E-NCX) of NCX current (I-NCX) and intracellular Ca2+ concentration ([Ca2+](i)),with the whole-cell voltage-clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted E-NCX were -73 and -11 mV at 3:1 and 4:1 stoichiometries, respectively. E-NCX measured were -69 +/- 2 mV (n = 11), -47 +/- 1 mV (n = 14) and -15 +/- 1 mV (n = 15) at holding potentials (HP) of -73, -42 and -11 mV, respectively. Thus, E-NCX almost coincided with HP, indicating that [Ca2+](i) and/or [Na+](i) changed due to I-NCX flow. Shifts of E-NCX (DeltaE(NCX)) were measured by changing [Ca2+](o) or [Na+](o). The measured values of DeltaE(NCX). were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo-1 fluorescence, [Ca2+](i) measured under the whole-cell voltage-clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca2+ accumulation, we inhibited I-NCX with Ni2+ and re-examined E-NCX during washing out Ni2+. With HP at predicted E-NCX at a 3:1 stoichiometry, E-NCX developed was close to predicted E-NCX and did not change with time. However, with HP at predicted E-NCX for a 4:1 stoichiometry, E-NCX developed initially near a predicted E-NCX for a 3:1 stoichiometry and shifted toward E-NCX for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.