We have examined the effect of butyrate on morphology, DNA synthesis, and epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. Butyrate added 2 h after plating retarded the flattening and maintained the polyhedral shape of the hepatocytes in culture. Both insulin- and EGF-stimulated DNA syntheses were slightly stimulated by butyrate at 1 mM but strongly inhibited at 5 mM. EGF receptor binding was also strongly affected by butyrate treatment of the hepatocytes. The freshly isolated hepatocytes (prior to plating) and the early-stage cultures (2 h) exhibited two classes of surface EGF receptors with high and low affinity (Kd approximately 0.05 and approximately 0.7 nM, respectively). With increasing time in culture there was a decrease in the total EGF receptor number and a corresponding reduction in the capacity for receptor-mediated EGF internalization. The high-affinity receptor class was more strongly reduced than the low-affinity class and was almost absent after 40 h in culture. Butyrate dose-dependently counteracted the decrease in the number of surface EGF receptors during culturing and preserved the high-affinity binding component. Thus, after 40 h, the cells cultured in the presence of butyrate (5 mM) had an approximately 50% elevation in the total number of receptors and the capacity to endocytose EGF compared to control cells, whereas the binding at low ligand concentration (0.02 nM) was increased 4-fold. The results suggest that butyrate, in addition to affecting morphology and DNA synthesis, also has marked effects on the hepatocyte EGF receptor status.