The effect of regucalcin, a calcium-binding protein isolaetd from rat liver cytosol, on (Ca²⁺-Mg²⁺)-adenosine triphosphatase (ATPase) activity in the plasma membranes of rat liver was investigated. ¹²⁵I-Regucalcin bound to the plasma membranes in the presence or absence of 0.1 mM Ca²⁺. Regucalcin (0.35 μM) increased the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity about 15% (p<0.01), and a higher concentration (4.0 μM) showed a remarkable effect. Also, regucalcin (0.35-2.0 μM) markedly increased the plasma membrane Mg²⁺-ATPase activity in the absence of Ca²⁺. The effect of regucalcin on (Ca²⁺-Mg²⁺)-ATPase activity was not regulated by the presence of guanosine-5'-O-(3-thiotriphosphate) (10^-5 and 10<-4>M), glucagon (10^-6 and 10^-5M) or norepinephrine (10^-7-10<-5>M), suggesting that the regucalcin effect is not linked to guanosine triphosphate (GTP)-binding protein in the plasma membranes. Vanadium (10 and 10 μM), which inhibits the phosphorylation of liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase, decreased the enzyme activity about 20% (p<0.01). This decrease was completely restored by the presence of regucalcin (1.0 μM). The present results indicate that regucalcin binds to the plasma membranes of liver cells and increases the (Ca²⁺-Mg²⁺)-ATPase activity. Regucalcin may be an activator of (Ca²⁺-Mg²⁺)-ATPase in the plasma membranes.