The involvement of serotonin and its receptor subtype in the induction of hepatocyte DNA synthesis was investigated in primary cultures of adult rat hepatocytes. Serotonin caused a dose-dependent increase in DNA synthesis in primary cultures of rat hepatocytes in the presence of epidermal growth factor (EGF) and insulin, as measured by [3H]thymidine incorporation. The serotonin S2receptor antagonists, ketanserin (10−6 mol/L) and spiperone (10−6 mol/L), blocked stimulation of DNA synthesis by serotonin. Displacement studies on [3H]5-hydroxytryptamine (5-HT) binding to crude membranes from control and regenerating liver tissue, using cold ketanserin and spiperone, showed an increased involvement of S2 receptors of serotonin in the regenerating liver during the DNA-synthetic phase. Serotonin enhanced the phosphorylation of a 40-kd substrate protein of protein kinase C (PKC) in the regenerating liver during the DNA synthetic phase of the hepatocyte cell cycle. This was blocked by ketanserin, indicating that serotonin S2receptor activates PKC, an important second messenger in cell growth and division, during rat liver regeneration. Our results show that serotonin can act as a potent hepatocyte comitogen and induce DNA synthesis in primary cultures of rat hepatocytes, which is suggested to be mediated through the serotonin S2 receptors of hepatocytes.