Oxaloacetate (OA), one of the substrates in the tricarboxylic acid (TCA) cycle, was found to induce proliferation of primary cultured rat hepatocytes. During the 5 days of culture in a serum-free medium with 30 mM OA, hepatocyte DNA synthesis had been induced to the same degree as by culturing with 10 ng/ml epidermal growth factor (EGF). A 3% mitotic index was observed in OA-treated cells 60-70 hr after plating. The OA-induced DNA synthesis was inhibited by transforming growth factor-beta in a dose-dependent manner and was abolished by 10 mM hydroxyurea. OA and EGF induced DNA synthesis synergistically with > 91% of the nuclei of the cells entering S phase at 48-72 hr in culture. Among other substrates in the TCA cycle, pyruvate, lactate, alpha-ketoglutarate, succinate, fumarate, and malate at equimolar concentrations to that of OA also induced DNA synthesis. However, their activities were about half or less than that with OA. These results show that OA is a hepatocyte mitogen in vitro.