To investigate the function of the RB protein, we have studied cellular RB binding proteins and protein kinases responsible for phosphorylation of the RB protein. (1) We purified a cellular RB-associated protein p56 which competes with SV40 large T antigen for binding to the RB protein. (2) In another experiment, we screened expression libraries of U937 monocytic leukemia cell line by West-Western method and obtained two cDNA clones that encode RB binding proteins. (3) The RB protein was found to be phosphorylated by cdk2 and MAP kinase in vitro. Most of the sites phosphorylated in vitro are the same as those phosphorylated in vivo and the time course of activation of cdk2 in the cell cycle were similar to that of phosphorylation of the RB protein.