We have recently shown that extensive proliferation of liver parenchymal cells takes place in adult male Xenopus frogs in response to estradiol-17 beta, which also induces synthesis and secretion of vitellogenin, the precursor of yolk proteins. We demonstrate here that liver parenchymal cells from adult male animals can be maintained for several weeks in a defined tissue culture medium containing added insulin, dexamethasone, and triiodothyronine. Under these conditions the cells do not divide, but can synthesize DNA. Maximum DNA synthesis occurs in cells that have achieved monolayer morphology under low plating densities. Estradiol-17 beta causes a dose-dependent increase in the number of cells synthesizing DNA, as well as inducing synthesis of vitellogenin. Estrogen-dependent, but not background, DNA synthesis is inhibited by the antiestrogen tamoxifen. These results imply that estradiol-17 beta acts directly on liver cells to initiate DNA replication, probably by interaction with a receptor protein and induction of specific gene transcription.