Mutations of the tumor suppressor gene p53 have been implicated in certain familial cases of breast cancer. We examined a series of 38 cases of nonfamilial bilateral breast cancer using antibodies CM1 and DO7 to p53 wild-type and mutant protein (Novocastra Laboratories) by the avidin-biotin-peroxidase complex method. The two antibodies reacted similarly. Mutant p53 protein was detected in 17 of 76 (22%) tumors but in only 3 of 38 (8%) paired tumors. There were no significant differences in p53 expression between synchronous (< 12 mos) and metachronous tumors (29% vs 17%, P = 0.09) or between first and second tumors (14% vs 26%, P = 0.29). Mutant p53 was detected bilaterally in one metachronous and two synchronous cases, which were amplified and sequenced and two synchronous cases, which were amplified and sequenced by polymerase chain reaction and single strand confirmation polymorphism. One synchronous case showed a bilateral mutation in exon 2-3; the other had a bilateral mutation in exon 8-9. In the metachronous case, a mutation could be demonstrated in only one breast. Analysis of all tumors demonstrated that when p53 protein is overexpressed in the first tumor, there is a 60% probability of overexpression in the second, whereas if absent from the first, it is unlikely to be present in the second. These data suggest that p53 mutations do not play a major role in the pathogenesis of bilateral disease in most women.