We have studied the effects of Pb2+ added in vitro on the movements of Ca2+ in renal cortical mitochondria and tissue slices. The isolated mitochondria rapidly accumulated 45Ca2+ at 25 degrees by a mechanism that was dependent on respiration and inhibited 96% by ruthenium red. A concentration of 10 microM Pb2+ inhibited the Ca2+ accumulation at least as effectively as did ruthenium red. About 20% of the Ca2+ accumulation persisted at 1 degrees with a similar sensitivity to inhibitors, including 60% inhibition by Pb2+. Similar results were obtained when the accumulation of Ca2+ at 25 degrees was measured by means of a calcium-sensitive electrode, Pb2+ inhibiting by 80%. Calcium that had been accumulated by mitochondria at 25 degrees was released completely by the ionophore A23187 or by 10 microM Pb2+. The release induced by Pb2+ was greatly inhibited by ruthenium red. The Ca2+ content of tissue slices of renal cortex increased 4-fold during incubation at 1 degree while the Ca2+ content of mitochondria within the slices more than doubled, the latter being determined by isolation of mitochondria from the slices after incubation. The presence of Pb2+ (200 microM) in the incubation medium of the slices substantially reduced the entry of Ca2+ into the whole slices and into mitochondria within the slices. When the slices preincubated at 1 degree were warmed to 25 degrees in oxygenated medium, they brought about a net extrusion of Ca2+, some of which was derived from the mitochondria; Pb2+ did not alter the final level of Ca2+ then attained in the slices, but it caused a significant decrease in the quantity retained in the mitochondria. We conclude that Pb2+ both inhibits the uptake of Ca2+ by renal cortical mitochondria and displaces Ca2+ from them, these effects occurring whether the mitochondria are isolated or in situ.