Loss of heterozygosity is commonly assumed to be due to deletion of the appropriate genomic region in one chromosome within a neoplastic cell but may be due to other mechanisms such as mitotic non-disjunction or somatic recombination leading to uniparental heterodisomy. We chose to study the genomic regions surrounding the p53 and RB1 tumor suppressor genes in breast carcinoma to evaluate the different mechanisms that could mediate loss of heterozygosity. A microsatellite analysis of polymorphic markers in 50 breast cancer samples showed loss of heterozygosity for at least 1 of the 10 markers analyzed in 50% of the tumors studied, and an overall 8.47% of the informative loci showed loss of heterozygosity. All of the cases with loss of heterozygosity were further analyzed for gene copy number of the tumor suppressor genes RB1 and p53 by fluorescence in situ hybridization of either tumor touch preparations or microdissected tumor nuclei with specific genomic probes. Surprisingly, all samples showed the presence of both copies of tumor suppressor genes, including 4/50 cases showing loss of heterozygosity of tumor suppressor gene-spanning markers. One of the 4 cases showed loss of heterozygosity of markers spanning a distance of 6 cM over the RB1 gene, with normal copy numbers of the gene. Three other cases showed loss of heterozygosity of markers within the tumor suppressor gene (RBI or p53) and at least one other spanning marker. No cases showed a simultaneous reduction to homozygosity of markers both near the tumor suppressor gene and distal loci. We suggest that the presence of both copies of the tumor suppressor gene in the cases with loss of heterozygosity of spanning markers and internal markers for that tumor suppressor gene could be explained by somatic recombination resulting in uniparental disomy, but not mitotic nondisjunction or deletion. As the mechanism for physical deletion of a chromosome may be different from those mediating somatic recombination, study of this phenomenon may identify different pathways of genomic instability that may be of diagnostic or treatment significance in breast or other cancers, particularly in those treatments based upon DNA-altering agents.