The interaction of various hormones and regucalcin on (Ca(2+)-Mg2+)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10(-6)-10(-4) M), phenylephrine (10(-6)-10(-4) M), and insulin (10(-8)-10(-7) M) in the reaction mixture produced a significant increase in (Ca(2+)-Mg7+)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin (3 x 10(-8)-3 x 10(-6) M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10(-4) M) which can inhibit the Ca(2+)-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 microM), isolated from rat liver cytosol, elevated significantly (Ca(2+)-Mg2+)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10(-4) M). The epinephrine (10(-5) M) or phenylephrine (10(-4) M)-induced increase in (Ca(2+)-Mg2+)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3 x 10(-6) M) was not weakened by the presence of regucalcin (0.5 microM). Moreover, GTP (10(-5) and 10(-4) M)-induced increase in (Ca(2+)-Mg2+)-ATPase activity was not seen in the presence of regucalcin (0.25 microM). The present finding suggests that the activating mechanism of regucalcin on (Ca(2+)-Mg2+)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.