Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 +/- 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium.