We have shown previously that a novel cell cycle-regulated histone H1 kinase activity, retinoblastoma kinase (RbK), associates with and phosphorylates the amino terminus of the Rb protein in G2-M. We have shown also that the amino terminus of p107, a Rb-related protein, does not associate with a similar kinase in vitro or in vivo. Here, we report that a RbK-like kinase associates with the amino terminus of p130, another Rb-related protein, only marginally. Moreover, the association of RbK with Rb in vitro is shown to require a discrete portion of the Rb amino terminus, amino acids 89-202. This region has been shown previously to be subject to inactivating mutations in retinoblastoma and to be required for Rb-mediated growth suppression in vitro. Taken together, these data indicate that the formation of Rb-RbK complexes may play an important role in Rb-mediated growth suppression. We have mapped two in vitro sites of Rb phosphorylation by RbK to sites that are phosphorylated in vivo and are targets of cyclin-dependent kinase phosphorylation in vitro. As such, at least some sites of RbK phosphorylation overlap with those of other proline-directed serine and threonine kinases. Consistent with this latter observation, we report that the trans-activation domain of c-myc is phosphorylated specifically by RbK in vitro at a site (serine 62) that is phosphorylated in vivo during G2-M, cell-cycle phases in which RbK activity is maximal.