We previously demonstrated that after transduction with the v-Ha-ras oncogene and grafting onto nude mouse hosts, primary epidermal keratinocytes with a null mutation in the p53 gene form tumors with increased growth rates and predisposition to malignant conversion relative to p53 wild-type keratinocytes (Weinberg WC, et al., Cancer Res 54:5584-5592, 1994). To further explore the cooperation between p53 loss of function and activation of the ras oncogene, cell lines were established from the normal epidermises of newborn and adult p53-null mice, and parallel subclones were reconstituted with the p53 val135 temperature-sensitive mutant. Reconstituted lines C, G, N, and V demonstrated functional p53 transcriptional activator activity at the wild-type-permissive temperature of 32Β°C, compared with the hygromycin-selected control line X and parental p53-null lines NHK4 and AK1b. Hygromycin-selected subclones, but not the parental lines, made normal skin in vivo; all cell lines made carcinomas after introduction of v-Ha-ras, independent of p53 status. These cell lines were compared in vitro at 32Β°C to maximize the amount of p53 val135 in the wild-type conformation. Expression of v-Ha-ras did not consistently alter p53-mediated transcriptional activity, suggesting that ras acts downstream or independently of p53. No correlation was observed between p53-mediated transcriptional activity and in vitro growth rates, colony formation after exposure to ultraviolet light, or suppression by normal neighboring keratinocytes. However, keratinocyte cell lines devoid of p53 and expressing v-Ha-ras formed colonies in soft agar; this was blocked at 32Β°C in all cell lines reconstituted with p53 val135 . These keratinocyte lines provide a model for exploring the role of p53 and the interaction of p53 and ras in keratinocyte transformation.