In accord with a set of prespecified principles of cell synchrony induction, a three-step procedure was developed to arrest cells reversibly in the G2 phase of the cell cycle. Cultures of Chinese hamster ovary (CHO) cells were presynchronized in early S phase by sequential treatment with isoleucine deficiency and hydroxyurea blockades; then they were switched to medium supplemented with either of two agents that inhibit DNA topoisomerase II activity by different mechanisms, Hoechst 33342 at 7.5 micrograms/ml for 12 hr or VM-26 at 0.5 micrograms/ml for 8 hr. Up to 95% of the cells accumulated in G2 phase under those conditions. After switch of Hoechst 33342-treated cells to drug-free medium, the cells divided as a highly synchronized cohort of cells within 3 hr. Up to 85% of the cells in a culture of human diploid dermal fibroblasts (HSF-55 cells) could be accumulated in G2 phase by placing cells presynchronized in early-S phase in medium containing Hoechst 33342 at 0.1 micrograms/ml for 10 hr. Reversal of G2 arrest in the HSF-55 cultures resulted in cells dividing synchronously over 3.5 hr. By varying the concentration of Hoechst 33342 and the duration of the treatment period, it was possible to alter the position within G2 phase at which cells accumulated. This synchronization protocol should greatly facilitate study of G2/M biochemical events in mammalian cells, in particular, those associated with cdc2 gene regulation of the onset of mitosis.