Hepatoma cells in culture exhibit a range of differentiated functions. Well differentiated hepatoma cells retain most of the functions of the adult liver, whereas dedifferentiated cells have lost most of them. In the present study, insulin binding and insulin effects were studied in four differentiated and two dedifferentiated cell lines derived from the Reuber hepatoma and in a partially differentiated cell line, HTC, derived from the Morris 7288C hepatoma. Specific insulin binding was lower in dedifferentiated cells than in partially differentiated and differentiated ones. In all cell lines, analysis of insulin binding yielded linear Scatchard plots. Although some variations in affinity of insulin for its receptor were observed, most of the differences in binding were accounted for by differences in insulin receptor number. In differentiated hepatoma cells, tyrosine aminotransferase (TAT)-specific activity was easily detectable, and insulin produced a 2- to 3-fold increase in enzyme activity within 4-6 h. By contrast, TAT activity in the dedifferentiated cells was low and did not respond to insulin. In the partially differentiated hepatoma HTC, insulin stimulated TAT only after basal TAT activity was induced by glucocorticoid treatment. Glycogen synthase (I and D forms) activities were detectable in all cell lines. In both differentiated and dedifferentiated Reuber hepatoma cells, insulin increased the I-form of glycogen synthase within 15 min. This effect of insulin was observed at lower insulin concentrations than stimulation of TAT activity. By contrast, insulin at any concentration was totally ineffective in stimulating glycogen synthase in glucocorticoid-treated and untreated HTC cells. These results indicate that in hepatoma cells, 1) insulin receptor number and insulin effect on TAT are modulated by the degree of cell differentiation; 2) the pathways of insulin action on TAT and glycogen synthase diverge in some postreceptor step(s) which is under independent control in differentiation; and 3) receptor and postreceptor defects exist in hepatoma cell lines which may be useful in dissecting the pathways of insulin action.