Summary DNA synthesis can be initiated in quiescent fetal rat hepatocytes in culture by the addition of dialyzed fetal calf serum, arginine, or ornithine with a time course similar to the one observed after partial hepatectomy of the rat. A lag period of 14-16 hr was observed between the addition of the stimulus to the cultures and the onset of DNA synthesis. Cells are committed to synthesize DNA after incubation with serum for 12 hr. Serum or ornithine is active only when incubated in conditioned medium. It is suggested that DNA synthesis in cultured fetal rat hepatocytes is regulated by intracellular levels of arginine and/or ornithine and/or their proximal metabolites. We gratefully acknowledge the assistance of M. Henahan, B. Raspenti, J. Rippon, L. White, and M. Williams. The work was supported by research grants from the American Cancer Society, California Division, The Damon Runyon Memorial Fund for Cancer Research, Inc., and by the National Institutes of Health, U. S., Public Health Service. Note added in proof: That arginine stimulates DNA synthesis in quiescent fetal rat liver cells in chemically defined medium has also been demonstrated by K. Koch and H. L. Leffert (submitted for publication), and H. L. Leffert and S. Sell (J. Cell Biol., in press).