The present study was designed to clarify the mechanism of cadmium (Cd)-induced toxicity in rat hepatocytes. Cd and Mg-ATP induced cellular acidification at concentrations lower than 25 microM. In Mg-ATP-treated cells, maximal acidification occurred within 2.5 min, with a subsequent return to control levels. In Cd-treated cells, maximal acidification (pH 6.76) occurred 10 min after exposure to the metal, then the cytoplasmic pH began to rise but did not return to normal. Cd eliminated the membrane potential of isolated mitochondria in media at both pH 6.5 and 7.4. This effect of Cd on membrane potential was approximately equivalent in both media when the metal concentration was 5 microM, but was more intense in the medium at pH 6.5 than in the medium at pH 7.4 at the metal concentration > 5 microM. Acidic medium alone had no effect on membrane potential. Mitochondrial uptake of Cd increased in a dose-dependent manner in media at both pH 7.4 and 6.5. The uptake of 5 microM Cd was significantly increased by acidic medium, however at Cd concentrations > 5 microM, there were no pH-dependent differences in Cd uptake. The incubation of hepatocytes in the medium at pH 6.0 and 6.5 resulted in 5 and 7.5% inhibition of mitochondrial respiration in intact cells, respectively. The presence of 10 microM Cd in the medium at pH 6.0 enhanced this inhibition. Mitochondrial respiration was inhibited to 60% of the control mitochondria at pH 7.4 when exposed to the medium at pH 6.5 without Cd and this inhibition was extended to 70% by the presence of 5 microM Cd. Cd in the medium at pH 7.4 had no deleterious effect on mitochondrial respiration at all concentrations examined. The results indicate that the respiratory activity of mitochondria is sensitive to the low pH rather than to Cd although the metal strongly inhibits the activity when in the medium at low pH. Therefore, it appears Cd-induced acidification plays an important role in the initiation of deteriorative processes in mitochondria.