Alterations of the p53 gene have been found in many human and animal tumors, and ras mutations have been seen somewhat less frequently. Loss of p53 tumor suppressor activity has been implicated in multistage tumor promotion/progression after initiation by Ha-ras or Ki-ras activation. Whether p53 or ras alterations occur during promotion of neoplastic transformation in JB6 cells has not been reported. Using a series of mouse JB6 variants representing different stages of progression toward the tumor-cell phenotype, we investigated whether mutational activation of Ha-ras and mutational inactivation and altered expression of the p53 gene occurred during progression. We report here that neither point mutations of the Ha-ras or p53 genes nor p53 structural alterations were involved in this process. Although there was no significant difference in steady-state levels of p53 mRNA, an elevated level of immunoprecipitable p53 protein was observed in a subset of transformed cells. We also report the detection of a second 2.2-kb p53-hybridizing transcript. This transcript was not translated into p53 protein and may be a nuclear precursor of the mature message. Both p53-hybridizing transcripts were downregulated by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (24 h) regardless of the stage of progression. We conclude from this study that in JB6 variants (1) mutational activation of Ha-ras and inactivation of p53 are unlikely to be involved in preneoplastic progression; (2) increased amounts of p53 protein may be involved in a subset of transformed cells, possibly reflecting a longer half-life, as demonstrated in other systems; and (3) late downregulation of p53 mRNA but not protein expression may be a secondary response in the TPA-mediated signal transduction pathway.