In isolated mitochondria extensive uptake of Ca2+ in the presence of an "inducing agent," e.g. inorganic phosphate (Pi), causes permeabilization of the mitochondrial inner membrane and a collapse of the mitochondrial membrane potential. In this study we tested whether the effect of phosphate occurs in intact hepatocytes. Rat hepatocytes were incubated with ATP to induce a sustained increase in intracellular Ca2+ ([Ca2+]i), dissipation of the mitochondrial membrane potential, and cell death (Zoeteweij, J. P., van de Water, B., de Bont, H. J. G. M., Mulder, G. J., and Nagelkerke, J. F. (1992) Biochem. J. 288, 207-213). Omission of Pi from the incubation medium delayed the loss of viability. The nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) had similar effects on [Ca2+]i and viability, but now omission of extracellular Pi completely protected against cytotoxicity. Exposure to ATP or ATP gamma S induced a large cellular uptake of Pi. With the use of video-microscopy a significant increase in mitochondrial free calcium was observed before the onset of cell death. Accumulation of mitochondrial calcium was reduced when extracellular Pi was omitted. These results suggest that, after induction of high [Ca2+]i by ATP in hepatocytes, 1) mitochondria accumulate calcium which is associated with cell toxicity and 2) intracellular Pi increases which stimulates mitochondrial calcium uptake. These observations support a calcium-dependent mitochondrial dysfunction, induced by phosphate, as a valid model for ATP-induced cytotoxicity in hepatocytes.