In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions. In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis. We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts. While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis. Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase. Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific. These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells. We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1.