Normal human breast epithelial cells were transfected with expression vectors containing the p53 gene mutated at either codon 143, 175, 248 or 273, or by infection with a recombinant retroviral vector containing the p53 gene mutated at codons 143, 175, 248, or 273. The breast epithelial cells were monitored for extension of in vitro lifespan and immortalization. Expression of some, but not all, p53 mutants resulted in an extension of in vitro lifespan. Experiments with the p53 temperature sensitive mutant 143ala revealed that at 32 degrees C, the nonpermissive temperature, the growth of breast epithelial cells was inhibited. At 37 degrees C, the mutant conformation, there was increased proliferation of cells, resulting in extension of in vitro lifespan. Breast epithelial cells expressing p53 mutant 273his maintained DNA binding and transcriptional activities and one clone immortalized after a period of growth arrest (crisis). The progression of this immortalization event was characterized by the reactivation of telomerase using the telomeric repeat amplification protocol (TRAP), and terminal restriction fragment analysis (TRF). This is the first reported immortalization of human mammary epithelial cells transfected with a mutant 53.