[Ca(2+)+Mg2+]-dependent ATPase activity in the pineal gland of the rat was examined. The enzyme possesses an apparent Km (Ca2+) of 0.23 microM, and moderately high affinity for Mg2+ and ATP (Km = 53.2 microM and Km = 86.4 microM, respectively). The ATPase activity is sensitive to low concentrations (I50 approximately 1 microM) of vanadate, which specifically inhibits Ca(2+)-ATPase in the plasma membranes of the erythrocyte, cardiomyocytes and synapses. The calmodulin antagonist trifluoperazine reduced significantly Ca(2+)-stimulated, Mg(2+)-dependent ATP hydrolysis. The [Ca(2+)+Mg2+]-dependent ATPase in rat pineal gland exhibits very high affinity for Ca2+, is highly vanadate sensitive and appears to require calmodulin. The enzyme is similar to the Ca(2+)-ATPase of the erythrocyte, cardiomyocytes and synaptic plasma membranes. These new findings may help to elucidate the mechanisms of intracellular calcium homeostasis and the effect of the enzyme on the synthesis of melatonin in the pineal gland.