Some of the progeny of isolated mouse embryo fibroblasts acquire the ability to grow indefinitely during cultivation, presumably through some mutational events. The relevance of p53 mutations and loss of heterozygosity to the mechanism of such immortal growth capability remains controversial. Since four bases in intron 1 of the p53 gene in C3H/HeJ mice are replaced by 13 different bases in DBA/2J mice, it is possible to distinguish maternal and paternal p53 alleles in the cells of F1 hybrids of these strains (C3D2F1) by electrophoresis of polymerase chain reaction fragments including the region. We established 23 spontaneously immortalized fibroblast cell lines from C3D2F1 mouse embryos and 29 transformed cell lines induced from one of the immortal cell lines, either by treatment with chemical carcinogens or by transfection with the c-Ha-ras gene. Of these 52 cell lines, only one, derived from fibroblasts unpassaged for 4 mo, showed p53 gene loss of heterozygosity and a structural alteration in the remaining allele. Our results demonstrated that p53 mutations are not a strict requirement for immortalization and transformation of mouse embryo fibroblasts in vitro.