Flow cytometric estimations of S-phase cells were carried out on cultures from three different cell lines and in frozen aliquots. A PCNA-extraction protocol was applied. Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA non-bound PCNA were compared with those obtained from total DNA histograms (Vindelöv and Christensen's technique, methanol-fixed whole cells and PCNA-extracted nuclei). No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells. Nevertheless, nuclei yield following PCNA extraction was highly variable, ranging from 63% to 10% (mean: 26%). In some cases, the extraction was not complete and samples had to be discarded. Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined. Because of these shortcomings, and the fact that is more costly and time consuming, the estimation of the S-phase fraction by means of bivariate DNA-bound PCNA/total DNA flow cytometric studies does not seem to surpass that obtained from standard DNA cell cycle analyses.