Flow cytometric cyclin expression/DNA content analysis, now commonly used, provides useful information on the mechanisms regulating cell cycle progression. However, this biparametric analysis does not make a clear‐cut distinction between G1 and S‐early or between S‐late and G2M phase cells. This paper proposes a new three‐parameter flow cytometric method with which to determine cyclin B1 levels in single cells in different cell cycle phases by coupling bromodeoxyuridine (BrdUrd) immunodetection and DNA content. DNA denaturation by HCl did not alter the level of cyclin B1. Differences in cyclin B1 expression were observed in seven human cancer cell lines of different origin. The percentage of cyclin B1‐positive cells and the cyclin B1 content per cell indicated different patterns. In some cases cyclin B1 accumulation preceded the G2M checkpoint, at which its content usually started to rise. Using available easily reproducible techniques, this flow cytometric approach gives details of intracellular variability in cyclin expression.  Cytometry 31:53–59, 1998. © 1998 Wiley‐Liss, Inc.