The p53 protein has become a subject of intense interest since the discovery that about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancer, suggesting a critical role for p53 protein in normal mammary epithelial cell (MEC) growth control. We previously demonstrated that abrogation of the p53 function by a cancer-derived p53 mutant, del239, was sufficient to induce immortalization of normal MECs. To further extend these findings and to examine the mechanism of mutant p53-induced immortalization of MECs, we tested the immortalizing ability of four selected p53 mutants (R175H, R248W, R249S, and R273H), which involve residues that cluster close to N239 in the three-dimensional structure and which are critical for the DNA-binding function of p53. Interestingly, two of these mutants (R175H and R249S) reproducibly immortalized 76N normal MECs, whereas the other two mutants (R248W and R273H) induced an extension of life span but not immortalization. These results further substantiate that selective ablation of p53 function with dominant-negative mutants is sufficient for immortalization of MECs. To determine whether abrogation of the transactivation function of endogenous p53 was important for the differential immortalizing ability of p53 mutants, we measured the effects of mutant p53 on the endogenous wild-type p53-mediated transactivation of a chloramphenicol acetyltransferase reporter linked to a consensus p53 binding DNA sequence in transiently transfected 76N MECs. All of the mutants, regardless of their immortalizing phenotype, abrogated the endogenous wild-type p53-mediated transactivation to a similar extent. Thus, abrogation of transactivation function is not sufficient for mutant p53-induced immortalization of normal MECs. The p53-immortalized MECs showed substantial telomerase activity; however, induction of telomerase activity occurred at late passages and was undetectable in mutant p53-expressing cells prior to immortalization. We suggest that mechanisms other than abrogation of transactivation and induction of telomerase activity determine the differential MEC-immortalizing behavior of various p53 mutants.