The hypothesis that ovarian cancer is multifocal in origin was examined using molecular genetic techniques. Patterns of allelic deletion on chromosome 17 were studied in 16 informative cases of Stage III serous epithelial ovarian carcinoma. DNA was extracted from specimens collected from the omentum and both ovaries, and the specific alleles and chromosomal loci involved in the deletion were identified and compared. In all cases, the patterns of allelic deletion were identical for the tumors that had been collected from different sites in the same patients. In addition, 4 of the 16 cases were heterozygous for the hypoxanthine phosphoribosyl transferase (HPRT) gene on the X-chromosome and were examined for methylation status. In all 4, the same parental allele of the HPRT gene was methylated in tumor cells collected from both ovarian and omental sites, suggesting that the patterns of inactivation of the X-chromosome are identical. This pattern of allelic deletion and HPRT-gene methylation in tumor samples collected from different sites implies that ovarian carcinomas have a unifocal origin.