Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a "burst" of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid "A" transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]