We have developed a "one-tube" triple staining procedure that allows the identification of intratumor phenotypic subpopulations by FCM. Solid tumors were dissociated by a combined mechanical/ enzymatic method. Ovarian ascites tumor cell aggregates were enzymatically dissociated using trypsin. An antikeratin 8/18 MAb was used to label the epithelial fraction of these tumor samples. A second MAb directed against the leukocyte common antigen (LCA) was applied to identify nonneoplastic DNA-diploid cells. Other MAbs used as a second marker were directed against a tumor-associate surface, a cytoplasmic, or a nuclear antigen. Cells were stained using subclass-specific fluorescein-isothiocyanate (FITC) or R-phycoerythrin (PE)-conjugated antibodies. DNA was stained with propidium iodide (PI). Triply stained samples were measured on a standard bench-top flow cytometer (FACScan). Keratin 8/18-positive cells, LCA-positive cells, and DNA could be simultaneously detected in dissociated breast carcinomas, mixed Müllerian tumors, and ovarian ascites specimen for refining DNA index (DI) calculations and S phase fraction (SPF) determination. Coefficients of variation (CV) of the G0G1 peak of the DNA histograms obtained ranged from 2.55% to 4.64% and from 2.71% to 4.71% for the DNA-diploid and -aneuploid fractions, respectively. In DNA-diploid tumors, antigen expression (HER-2/Neu, proliferating cell nuclear antigen) could be analyzed without interference of fluorescence signals from nonneoplastic cells. Neoplastic tumor subpopulations were clearly identified based on both DNA-ploidy status and heterogeneity of antigen expression. The present method offers new possibilities for multiparameter DNA FCM on clinical samples and enables the identification of intratumor neoplastic subpopulations based on antigen expression and DNA-ploidy status.