Bivariate flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was performed on prostate carcinoma cell lines (PC-3, DU-145). For both cell lines 100% methanol fixation provided optimal fluorescence intensity of PCNA. The ratio of PCNA/DNA increased in late G1 through early S/phase, followed by a decrease in mid- and late S and enhancement in G2/M phase. PCNA expression was increased in G2/M phase cells treated for 48 h with vinblastine. A slight decrease in PCNA expression was observed with cyclohexamide treatment. Hydroxyurea induced an increase in S-phase fraction along with enhanced PCNA expression. Methotrexate and Adriamycin had little effect on the cell cycle compartments of PC-3 or DU-145; however, methotrexate decreased PCNA expression, while Adriamycin enhanced it. Cisplatin increased S-phase in both cell lines, increasing PCNA expression in PC-3 and decreasing it in DU-145 cells. The data on the effects of drug treatment point to a dissociation between PCNA expression and S-phase fraction as calculated from the DNA distribution. In some cases, e.g., the cisplatin studies, different effects were obtained in the two different cell lines treated with the same drugs. Whether changes in PCNA expression will provide more useful information than S-phase fraction for evaluation of potential antitumor drugs is not known.