The study presented here was designed t o further investigate the role o f transforming growth factor-a (TGFa) in skin tumor promotion by examining the ability of 12-O-tetradecanoylphorbol-l3-acetate (TPA) and several non-phorbol ester promoters t o alter TGFa mRNA and protein levels in mouse epidermis. Total RNA was isolated from SENCAR mouse epidermis at various times after single topical treatments with TPA (3.4 nmol), chrysarobin (220 nmol), okadaic acid (2.5 nmol), and thapsigargin (8.5 nmol). Northern analyses of these isolated RNA samples revealed that all four tumor promoters transiently elevated TGFa mRNA levels. Whereas TPA, okadaic acid, and thapsigarin elevated TGFamRNA levels over similar time courses (peak at 4-8 h), chrysarobin elevated TGFa mRNA levels with a markedly delayed time course (peak at 24-48 h). More detailed studies with TPA also revealed that multiple treatments (four over a 2-wk period) transiently elevated TGFa mRNA in both the epidermis and the dermis. The time courses for changes in TGFa mRNA after multiple TPA treatments were similar for both tissues. To facilitate studies of altered TGFa mRNA expression in mouse epidermis and possibly other mouse tissues, a semiquantitative reverse transcriptase-polymerase chain reaction method was developed. This method faithfully revealed changes in TGFa mRNA levels with all four tumorpromoting agents similar t o those determined by northern blot analyses. lmmunofluorescence analysis of frozen sections from promoter-treated skin revealed elevated TGFa protein levels in both epidermis and dermis, although staining was most intense in the epidermal layer. lmmunofluorescence analysis o f epidermal hyperplasia adjacent t o a full-thickness wound also demonstrated significant epidermal TGFa staining. Collectively, these results indicate that mechanistically diverse tumor promoter stimuli elevate TGFa mRNA and protein in SENCAR mouse epidermis. Elevated levels of TGFa may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli.