Di(2-ethylhexyl)phthalate (DEHP), a confirmed promoter of hepatocellular carcinogenesis in mice, was studied for tumor-promoting activity on the skin of SENCAR mice in vivo and on JB6 mouse epidermal cells in vitro. DEHP enhanced transformation to anchorage-independent growth of promotable JB6 clonal lines, C141, C121, and R219. DEHP induced colony formation in concentrations from 500 p.p.m. to 20 000 p.p.m./ml culture medium with maximum induction occurring at 10 000 p.p.m./ml. DEHP was inactive as a complete promoter of skin carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) in SENCAR mice. Like the plant-derived natural product mezerein, however, DEHP significantly enhanced skin carcinogenesis in SENCAR mice when initiation by DMBA was followed by short term applications (2x/week, 2 weeks) of 12-O-tetradecanoylphorbol-13-acetate prior to application of DEHP. A random sample of the tumors observed was processed and examined histologically. Most neoplasms were papillomas; a few were squamous cell carcinomas. The kinds of tumors seen were identical with those reported in two-stage carcinogenesis experiments in SENCAR mice in which phorbol ester tumor promoters have been employed. Thus, DEHP promotes transformation in JB6 mouse epidermal cells and acts as a second-stage promoter in mouse skin carcinogenesis. DEHP therefore has promoting capability for at least two distinct kinds of epithelium in mice.