The cyclins and their catalytic partners, the Cyclin Dependent Kinases (CDKs), are essential for progression through the cell cycle. Cyclin/kinase complexes containing cyclins A or E are active primarily in late G1 to S phase and both have been shown to phosphorylate histone H1 and the retinoblastoma gene product (pRb) in vitro. Despite these similarities, cyclins A and E display differences in CDK activation and substrate specificity. We find that in vitro, cyclin E/CDK2 and cyclin A/CDK2 phosphorylate histone H1 similarly but only cyclin A/CDK2 phosphorylates lamin B. While both cyclin A and cyclin E bind CDK1 efficiently, only cyclin A activates CDK1 kinase activity. Using chimeric proteins between cyclins A and E we find that both the cyclin box and C-terminus of cyclins A and E are required for CDK binding, activation and targeting of substrate specificity.