The retinoblastoma tumor suppressor protein, pRB, is inactivated by phosphorylation. While existing evidence is strong that such phosphorylation is mediated by one or more cyclin-dependent kinases (CDKs) active during G 1 /S, it remains unclear which of the various CDKs is responsible. We show here that three candidate pRBinactivating kinases, CDK4-cyclin D1, CDK2-cyclin E, and CDK2-cyclin A, phosphorylate pRB differentially, each on a subset of authentic pRB phosphorylation sites. Notably, two neighboring pRB phosphate acceptors, threonine 821 and threonine 826, which have previously been implicated in the regulation of LXCXE protein binding, are phosphorylated by different CDKs. We demonstrate that phosphorylation by either CDK2-cyclin A, which phosphorylates T821, or CDK4-cyclin D1, which phosphorylates threonine 826, can disable pRB for subsequent binding of an LXCXE protein. However, only one of these two kinases, CDK2-cyclin A, can dissociate a pre-existing LXCXE protein-pRB complex. We provide evidence that prior binding of an LXCXE protein blocks access to certain residues specifically targeted by CDK4-cyclin D1, explaining the inability of this kinase to resolve such complexes. While these results are not direct proof of the relevance of differential pRB phosphorylation in cells, our findings support a model whereby full phosphorylation of pRB may require the action of more than one kinase and explains how such differential phosphorylation by different CDKs might translate into a differential regulation of downstream effector pathways.The retinoblastoma tumor suppressor gene encodes a nuclear phosphoprotein, pRB, which plays a central role in control of the cell cycle. pRB seems to act as a check during the G 1 phase of the cell cycle, ensuring that cells do not enter S phase until such a time as is appropriate. In some differentiating cells this function is extended, with pRB mediating their permanent exit from the cell cycle (see review in Ref. 1). These functions of pRB seem to rely on its ability to bind to, and alter the activity of, transcription factors including E2F-1, PU.1, ATF-2, UBF, Elf-1, MyoD, and BRG-1 (reviewed in Ref. 2). pRB appears to inhibit transcription from genes necessary for cell cycle progression by binding to certain of these transcription factors (the best studied of which is E2F-1) while promoting transcription from differentiation-specific genes through binding others (e.g. MyoD).Hyperphosphorylation of pRB is thought to inactivate its functioning by disabling its transcription factor-binding abilities, thus allowing progression of cells through the division cycle (3). The likely mediators of this phosphorylation are members of the family of proline-directed, serine/threonine, cyclindependent kinases (CDKs) 1 ; they phosphorylate authentic sites on pRB and are themselves thought to play a key role in cell cycle control (4, 5).Hyperphosphorylation of pRB is accompanied by release of pRB from nuclear tethering and a change in pRB's mobility in SDS-containing gels (6 -10, 29)....