Intratumor phenotypic heterogeneity is one of the characteristics of breast carcinomas, and genetic mechanisms are likely to contribute to it. We have studied breast cancer clonal heterogeneity by cytogenetic analysis of multiple tumor samples (one from each tumor quadrant) as well as samples of macroscopically normal surrounding breast tissue from 3 patients with this disease. Clonal chromosome aberrations were found in all 8 successfully analyzed samples from the carcinomas. Two to 6 cytogenetically unrelated clones were detected in each case, unevenly distributed among the tumor quadrants. Karyotypic abnormalities were also found in 4 out of 9 macroscopically tumor-free samples from the surrounding tissue; in 2 of these samples, a ductal carcinoma in situ was detected histologically, and the cytogenetic evidence suggests that the remaining 2 samples also contained neoplastic cells. Quantitative analysis of the findings revealed a statistically significant higher frequency of karyotypically abnormal cells in samples with a histologic diagnosis of carcinoma vs. samples without any detected malignancy. That cells bearing cytogenetic evidence that they belong to the tumor parenchyma are left behind during breast-conserving surgery for carcinoma of the breast may account for the relatively high long-term local relapse rates seen in this disease.