The two-stage murine skin tumorigenesis model is widely used to study the development of squamous cell neoplasias. We have investigated expression of the p53 and retinoblastoma tumor suppressor genes in eight murine skin tumor cell lines of varied histopathology and malignant potential, in seven in vivo-derived clones from these cell lines, and in 39 primary short-term cultures of similarly induced skin tumors at various stages of tumor progression. One squamous cell carcinoma cell line and three more malignant clones derived from it revealed mutations of the p53 protein by immunoprecipitation analyses despite normal-sized p53 transcripts. Sequence analysis identified the nature of the point mutations in these lines, a G→C transversion in codon 132. Mouse retinoblastoma transcripts and protein were unaltered in all the cell lines examined. Among short-term cultures of skin tumors, the p53 gene appeared normal in all papillomas and early well-differentiated carcinomas by Southern and immunoprecipitation analyses. In contrast, four of eight tumors from later stages of promotion (50–60 weeks) possessed alterations in p53, including loss of the p53 product, and loss of immunoreactivity with a murine-specific antibody recognizing only wild-type p53 protein. Loss of heterozygosity at the p53 locus was similarly observed in several more malignant tumors from later stages of promotion. In contrast retinoblastoma expression was normal regardless of the stage of promotion or histological grade of the tumor. Direct sequence analyses of exons 5 through 8 of the p53 gene in eight advanced murine skin tumors revealed a 25% incidence of p53 mutations. These point mutations were located in codons 245 and 263. Collectively, these data indicate that alterations in the p53 gene occur in 25 to 50% of murine skin tumors induced by the two-stage tumorigenesis protocol and are later events in murine skin tumor progression. Moreover, these alterations are associated with tumors possessing a more malignant and/or poorly differentiated phenotype.