Cyclins A and B1 activate cyclin-dependent kinases CDK2 and CDC2, which regulate cell progression through S and G2. Expression of these cyclins is generally measured in populations of synchronized cells, by immunoblotting. Such studies neither provide information regarding intercellular variability in cyclin expression nor yield precise data on a time relationship between initiation and termination of DNA replication in relation to cyclin expression. Furthermore, cell synchronization by DNA polymerase inhibitors or excess of thymidine induces cell growth imbalance and alters expression of cyclins, thereby introducing an experimental bias. Using a novel flow cytometric method of detection of incorporated bromodeoxyuridine (BrdUrd) in the present study, we have been able to correlate expression of immunocytochemically discerned cyclins A and B1 with incorporation of BrdUrd and the cell cycle position of individual MOLT-4 cells. On the basis of differences in amount of incorporated BrdUrd and DNA content, the following cohorts of cells in narrow windows of the cell cycle were identified: (a) cells initiating and (b) terminating DNA replication during a 1-h pulse of BrdUrd; (c) cells replicating DNA throughout the duration of BrdUrd pulse; (d) G1 cells; and (e) G2 cells that remained in G2 for at least 1 h after exiting S phase. These populations were characterized with respect to expression of cyclins A and B1. Expression of cyclin A was an early event of S phase, and 84% of cells entering S phase during 1 h of exposure to BrdUrd were already cyclin A positive. More than 95% of S-phase cells, as well as the cells exiting S during BrdUrd pulse, were also cyclin A positive. The maximal rate of accumulation of cyclin A was seen during the first hour of progression through S phase. In contrast, the maximal accumulation rate of cyclin B1 showed cells during the first hour of progression through G2. A strong correlation between expression of cyclin A and the rate of DNA replication, estimated by the degree of BrdUrd incorporation (r = 0.99), was observed.