In a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 mum. The half maximal transport rate occurred at 34 mum Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of the ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.