We previously reported that tumor necrosis factor-alpha (TNF-alpha) increases in vivo hepatocyte mitoses and liver regeneration in rats. In the present studies, we used in vitro hepatocyte cultures to determine whether TNF-alpha itself was mitogenic or whether other cytokines (IL-1 beta and IL-6) that share similar actions with TNF-alpha might be involved. Hepatocytes were cultured with TNF-alpha (4, 40 or 400 U/ml), IL-1 beta (0.1, 1 and 10 ng/ml) or IL-6 (0.2, 2, or 20 ng/ml) for 24 h and 3 d. Incorporation of [3H]thymidine was increased significantly by TNF-alpha (40 and 400 U/ml) at 3 d. Both IL-1 beta and IL-6 at all concentrations significantly decreased [3H]thymidine incorporation at 3 d. These results indicate that TNF-alpha has a direct mitogenic action on hepatocytes. In contrast, IL-1 beta and IL-6 appear to suppress DNA synthesis in hepatocytes.