Flow cytometric DNA content measurements have demonstrated extensive DNA ploidy heterogeneity in primary breast carcinomas. However, little is known at the molecular level about the clonal relationship between these tumor cell subpopulations, or about the molecular genetic changes associated with aneuploidization. We have used flow cytometric cell sorting to dissect some of this complexity by isolating clonal subpopulations in breast carcinomas for comparative molecular genetic analysis. Clonal subpopulations were isolated from 12 primary breast carcinomas and 5 lymph node metastases from 4 cases based on DNA content and cytokeratin 8/18 labeling. DNA from these clones was screened for allelic imbalances with 92 polymorphic microsatellite markers mapped to 39 different chromosome arms. Diploid and aneuploid populations were concurrently present in 11 out of 12 primary tumors. The DNA ploidy status of primary tumors was identical to that of the related lymph node metastases. Allelic imbalance was present in 10 out of 11 diploid clones (mean, 3.4 +/- 4.2). All allelic imbalances observed in the diploid clones recurred in the cognate aneuploid clones, but were, in the latter, accompanied by additional allelic imbalances at other loci and/or chromosome arms (mean, 10.9 +/- 5.8). In only two of the four metastatic cases did the allelotypes of metastatic clones show small differences relative to their cognate primary tumors. The primary diploid tumor clone recurred in all lymph node metastases. This study indicates that the majority of allelic imbalances in breast carcinomas are established during generation of DNA ploidy diversity. Recurrence of the allelic imbalances in diploid clones in the aneuploid clones suggests linear tumor progression, whereas the simultaneous presence of early diploid and advanced aneuploid clones in both primary and metastatic tumor sites suggests that acquisition of metastatic propensity can be an early event in the genetic progression of breast cancer.