The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the transcription factor E2F and reversed the Rb G1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.