The effect of activin-A on glycogenolysis was studied in isolated rat hepatocytes. Activin-A stimulated glucose output in hepatocytes in a dose-dependent manner. The maximal effect of the glycogenolytic action of activin-A, which was about 50% of the glucagon action, was obtained at 10(-9) M. When 10(-9) M activin-A and 5 x 10(-9) M glucagon were added simultaneously, the actions of these two agents were additive. In contrast, there was no additivity when 10(-9) M activin-A and 10(-8) M angiotensin-II were added. Activin-A did not increase cAMP at any doses tested, but induced a rapid increase in cytoplasmic free calcium concentration. Activin-A increased the cytoplasmic free calcium concentration even in the presence of 1 microM extracellular calcium, suggesting that activin-A caused calcium release from an intracellular calcium pool(s). The internal calcium pool affected by activin-A appeared to be the same as that affected by either angiotensin-II or vasopressin. When [3H] inositol-labeled hepatocytes were incubated with activin-A, radioactivity in the inositol trisphosphate fraction was rapidly increased. These results indicate that activin-A acts on rat hepatocytes and stimulates glycogenolysis by activating the calcium messenger system.