1. Prior treatment of rats with certain steroid hormones affects the binding of calcium to subsequently isolated liver mitochondria in vitro. Mitochondria isolated from animals treated with cortisol bind less calcium than do mitochondria from control animals under conditions in which binding is supported either by adenosine triphosphate alone or primarily by a respiratory substrate. Treatment with other glucocorticoids (prednisone and cortisone) likewise diminishes substrate-dependent calcium binding to mitochondria in vitro. 2. Prior treatment with deoxycorticosterone enhances the binding of calcium to subsequently isolated mitochondria in both ATP- and substrate-dependent systems. Treatment with aldosterone and 17β-estradiol also increases calcium binding under respiratory substrate-dependent conditions in vitro. Treatment with testosterone or progesterone is ineffective in altering mitochondrial calcium binding in vitro. 3. The results suggest that treatment of rats with cortisol or deoxycorticosterone affects the ATPase activity of subsequently isolated mitochondria and that this effect may be responsible for alterations in the ATP-dependent binding of calcium in vitro. The effect of cortisol treatment on diminishing ATP-dependent calcium binding is potentiated by the addition of oligomycin in vitro. 4. Antimycin inhibits the substrate-dependent binding of calcium to mitochondria in vitro. This effect is potentiated by cortisol treatment of the animal, probably by virtue of a steroid-induced block in the electron transport sequence at or near the antimycin-sensitive step. 5. It is suggested that cortisol treatment interferes with the utilization of ATP or respiratory substrate necessary to support mitochondrial calcium binding in vitro.