Serum-free medium conditioned by confluent cultures of JM1 or JM2 rat hepatocellular carcinoma cells stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. The hepatotrophic activity was nondialyzable in Mr 50,000 cutoff membranes, heat (60C) and acid stable, and sensitive to trypsin and dithiothreitol treatment. Gel filtration of concentrated JM1 or JM2 conditioned medium on Sephadex G-100 separated the activity into two regions, the major broad peak migrating with apparent Mr 25,000. Chromatography fractions active in the hepatocyte proliferation bioassay also inhibited specific binding of iodinated epidermal growth factor to cultures of A431 carcinoma cells and rat hepatocytes. These results suggest that neoplastic liver cells synthesize and secrete polypeptide growth factors which can bind to epidermal growth factor receptors and stimulate proliferation of normal adult rat hepatocytes in primary culture. (Copyright (c) Cancer Research Vol. 45, December 1985.)