Cells from pleural effusions (28 malignant and 14 nonmalignant with reactive mesothelial cells) were examined in parallel by means of conventional cytology and multiparametric flow cytometry. The latter included the evaluation of DNA ploidy, the calculation of DNA index (DI), the determination of S phase fraction (SPF), and cell proliferation associated with Ki-67 antigen expression. Propidium iodide was used for the DNA staining, and fluorescein-conjugated monoclonal mouse antibodies were applied to the human Ki-67 antigen staining. The specimens were analyzed by the Becton Dickinson (BD) FACScan apparatus. 10(4) events were collected, and thereafter the obtained data were analyzed by the software Lysis II and CellFit (BD) for DNA and Ki-67 data analysis, respectively. Out of the 28 cases examined, 18 (64%) of the malignant effusions were aneuploid. All of the benign effusions were typically diploid. The average SPF of benign, malignant diploid, and malignant aneuploid cases was 6, 6, and 30 respectively. The comparison between SPF and DI in malignant cases showed a significant correlation (p < 0.001). The average of Ki-67-positive cells that were benign, malignant diploid, and malignant aneuploid were 11, 19, and 35, respectively. There was a significant correlation between Ki-67 expression, SPF (p < 0.05), and the percent of the aneuploid cell population (p = 0.01). The sensitivity of conventional cytology, the determination of DNA ploidy and of Ki-67 expression, was 80, 64, and 92%, respectively; specificity was 100% for cytology and DNA ploidy and 46% for Ki-67 expression. These data show that Ki-67 antigen expression is a potentially useful adjunct to cytometric DNA content analysis, determination of SPF, and conventional cytology in the diagnosis of malignant pleural effusions.