We determined whether hepatic stimulator substance shares its mitogenic specificity for hepatocytes with nonparenchymal epithelial cells in the hepatocyte lineage. Cell lines designated HTC (derived from a rat hepatoma known to respond to hepatic stimulator substance) and FNRL, K-16 and K-22 (derived from rat liver nonparenchymal epithelial cells) were used. After exposure to hepatic stimulator substance, [3Hlthymidine incorporation into DNA was significantly increased (p < 0.001) in HTC, FNRL and K-16 cells, but not in K-22 cells. Fluorescence-activated cell sorting demonstrated that the mitogenic response to hepatic stimulator substance was associated with a greater proportion of cells entering the S phase. Epidermal growth factor, alone or in combination with hepatic stimulator substance, had no significant mitogenic effect on FNRL cells, but exposure of these cells to transforming growth factor-pl inhibited [3H]thymidine incorporation into DNA and reduced the proportion of cells in the S and G2/M phases. Simultaneous exposure of FNRL cells to hepatic stimulator substance and transforming growth factor-61 abrogated the inhibitory effect of transforming growth factor-pl. Comparison of butyrate-synchronized HTC cells with hepatic stimulator substance-treated HTC cells showed that S-phase progression in these conditions was different, with no intervening cell cycle arrest after treatment with hepatic stimulator substance. Mitogenic stimulation of FNRL and K-16 cells with the liver-specific growth factor hepatic stimulator substance suggests that these cells are of hepatocyte lineage. These results strengthen the evidence for a possible link between hepatocytes and nonparenchymal liver epithelial cells during liver biogenesis and differentiation. (HEPATOLOGY 1992; 15:485-491.)