Critical cell cycle transitions are controlled by the coordinate actions of the p34cdc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc28-4 mutation in Saccharomyces cerevisiae using a lambda YES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.