The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.