TG.AC transgenic mice harbor a v-Ha-ras transgene and retain two normal c-Ha-ras alleles and are susceptible to skin tumor formation by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether normal c-Ha-ras antagonizes the oncogenic potential of the v-Ha-ras transgene and/or whether additional non-Ha-ras 7,12-dimethylbenz(a)anthracene (DMBA) initiation target genes exist in mouse skin, which could cooperate with v-Ha-ras to increase the frequency of initiation, rate of promotion, or risk of malignant conversion, we treated TG.AC mouse skin with a single subthreshold dose of DMBA. This was followed by limited TPA or diacylglycerol promotion to select for cells with additional genetic alterations over those cells containing the v-Ha-ras transgene only. DMBA-treated/TPA-promoted TG.AC mice demonstrated a 10-fold increase in the average number of papillomas per mouse, a greater incidence of papilloma bearing-mice, and an increased papilloma growth rate when compared to acetone-treated/TPA-promoted TG.AC mice. These profound changes in papilloma frequency and growth occurred in the absence of the characteristic DMBA-induced A182-->T mutation in c-Ha-ras and immunohistochemical nuclear staining for p53 protein. DMBA-treated/acetone-promoted TG.AC mice did not develop any tumors. Limited promotion with the model diacylglycerol, sn-1,2-didecanoylglycerol, similarly produced an average of 10-fold more papillomas in DMBA-treated mice than in acetone-treated/sn-1,2-didecanoylglycerol-promoted TG.AC mice. DMBA-treated/TPA-promoted TG.AC mice developed their first malignancy by 16 weeks, and by 30 weeks, 50% of the mice developed malignancies, whereas no malignancies were observed in acetone-treated/TPA-promoted TG.AC mice. These results indicate that there exist unidentified DMBA initiation target genes in TG.AC mouse skin that cooperate with mutant Ha-ras to increase papilloma frequency, growth, and malignant conversion, and that promoter treatment can influence malignant conversion by selecting for cells with multiple genetic alterations.