The effects of tumor-promoting phorbol diesters on ornithine decarboxyalse (ODC) activity and DNA synthesis in normal and chemically transformed hamster embryo fibroblasts (HEF) in culture were studied. Only those phorbol diesters with promoting activity in mouse skin induced ODC in HEF. ODC was induced in both cell types by 12-O-tetradecanoyl-phorbol-13-acetate (TPA); maximal induction occurred 4 to 6 hr after the addition of the promoter to the medium of confluent cultures and was greater in transformed cells than in normal cells. The extent of induction in transformed cells treated with 0.016 to 1.6 micron TPA was dose dependent. The cellular concentrations of the polyamines, particularly putrescine, also increased after TPA treatment. The addition of TPA to confluent cultures of either normal or transformed HEF did not produce an increase in cell number or the percentage of [3H]thymidine- labeled nuclei and did not stimulate the incorporation of [3H]thymidine. ODC also was induced by adding fresh medium to the cultures. When both fresh medium and TPA were added, the effect of the medium was markedly potentiated in transformed, but not in normal, cells. These experiments demonstrate that tumor promoters specifically induce ODC in HEF without increasing the rate of DNA synthesis and that normal and transformed HEF differ in the levels of ODC activity attained after exposure to promoters.