Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.