A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay-positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense-type mutations or missense mutations that may affect 3D-structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow-up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease-free (recurrence-free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease-free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow-up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients.