To better understand the progression of epithelial cells from a normal to a malignant phenotype, we have compared various responses of normal, preneoplastic, and neoplastic hamster epidermal cells to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA, but not fresh serum-containing medium, induced ornithine decarboxylase (ODC) activity in primary hamster epidermal cells; the combination of TPA and medium was no more effective than was TPA alone. In four epidermal cell lines derived after treatment of cultures with carcinogen, the pattern of ODC induction at low passage levels was similar to that observed in primary cells. However, by the time the cell lines had become anchorage independent or formed tumors in appropriate hosts, the effect of TPA and fresh medium on ODC activity was synergistic. TPA did not affect the growth rate, plateau density, cellular morphology, or size of either primary epidermal cultures or continuous cell lines. Incorporation of [3H]thymidine by primary epidermal cells and some, but not all, cell lines was stimulated by addition of fresh medium but inhibited by TPA alone. In combination with fresh medium, however, TPA potentiated [3H]thymidine incorporation. We conclude that hamster epidermal cell lines acquire a characteristic synergistic induction of ODC activity by TPA and fresh medium as they progress toward malignancy. However, no consistent alteration is observed with respect to the parameters of mitogenesis examined.