The effect of Ca 2ϩ -binding protein regucalcin on Ca 2ϩ -ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 M) in the enzyme reaction mixture led to a significant increase in Ca 2ϩ -ATPase activity. Regucalcin significantly stimulated ATP-dependent 45 Ca 2ϩ uptake by the mitochondria. Ruthenium red (10 Ϫ5 M) or lanthanum chloride (10 Ϫ4 M), an inhibitor of mitochondrial Ca 2ϩ uptake, completely inhibited regucalcin (0.25 M)-increased mitochondrial Ca 2ϩ -ATPase activity and 45 Ca 2ϩ uptake. The effect of regucalcin (0.25 M) in increasing Ca 2ϩ -ATPase activity was completely inhibited by the presence of digitonin (10 Ϫ2 %), a solubilizing reagent of membranous lipids, or vanadate (10 Ϫ5 M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 M) on Ca 2ϩ -ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 M), a modulator protein of Ca 2ϩ action that could increase mitochondrial Ca 2ϩ -ATPase activity. The present study demonstrates that regucalcin can stimulate Ca 2ϩ pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca 2ϩ -ATPase.