We have previously demonstrated that injection of adult male frogs with estradiol-17 beta causes extensive proliferation of liver parenchymal cells together with the induction of vitellogenin (R. J. Spolski, W. Schneider, and L. J. Wangh (1985) Dev. Biol. 108, 332-340). In addition, purified parenchymal cells placed in culture synthesize DNA in an estrogen-dependent manner (B. S. Aprison, L. Martin-Morris, R. J. Spolski, and L. J. Wangh (1986) In Vitro 22, 457-464). We now describe conditions under which secondary exposure to estradiol-17 beta, either in vivo or in vitro, can lead to further DNA synthesis and cell division. The extent of this proliferation depends upon both the magnitude of the primary dose and the length of time elapse before secondary stimulation. A hormone dose of 0.5 mg, which causes little cell proliferation initially, allows for maximal secondary proliferation in response to 2.0 mg, while a maximal primary dose of 2.0 mg substantially inhibits further division in response to a secondary treatment with the same hormone dose. Cell culture experiments demonstrate that the failure of liver cells, in maximally stimulated males, to synthesize DNA in response to estrogen is not irreversible. But, cell crowding in culture does restrict DNA synthesis. The restrictions seen in vivo may therefore be due to structural features of the intact tissue rather than to terminal differentiation at the genetic level. These results are discussed with regard to our understanding of hormone-dependent differentiation in the frog liver system.