A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9 insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links growth factor stimulation with the onset of cell cycle progression.