Proline-directed protein kinase (PDPK) is characterized as a cytoplasmic oncogenic serine/threonine kinase that is activated by growth factor-mediated mechanisms and is proposed to function in mammalian somatic cells as an S phase promoting factor. The present study was undertaken to assess the hypothesis that p34cdc2/p58cyclinA PDPK is a physiologically relevant form of the p34cdc2 protein kinase that phosphorylates and inactivates the product of the retinoblastoma/osteosarcoma tumor susceptibility gene (Rb protein). In the course of these studies it was determined (fortuitously) that the p34cdc2/p58cyclinA PDPK purified from the cytosol of FM3A mouse mammary carcinoma cells was 'contaminated' by several high molecular weight substrate proteins that essentially co-purified with the protein kinase, one of which was identified as the Rb protein itself (p105Rb). High-resolution fast protein liquid chromatography (FPLC) revealed that the Rb protein co-purified with a particular subset of the PDPK heterodimer, i.e. with a single species of the 58 kDa cyclinA doublet. The subset of PDPK associated with the Rb protein exhibited somewhat lower specific enzyme activity, as judged by in vitro kinase assays and comparative Western blotting. Immunoprecipitation studies confirmed that p105Rb is physically associated with the p34cdc2/p58cyclin A PDPK. Further studies confirmed that the underphosphorylated Rb protein (p105Rb) present in G1 lysates of synchronized human MG63 osteosarcoma cells could be readily phosphorylated by purified PDPK in vitro, resulting in the characteristic shift in the apparent molecular mass (SDS-PAGE) of the Rb protein that is reported to accompany the hyperphosphorylation and functional inactivation of this protein. Moreover, the induction of the cyclin A subunit of PDPK in these synchronized MG63 cells was found to be closely correlated with the cell cycle-dependent phosphorylation of the Rb protein. From these studies it is concluded that the growth factor-sensitive PDPK is a physiological Rb kinase, which may function to inactivate the Rb protein in vivo.