The retinoblastoma tumor suppressor gene (Rb) has many functions within the cell including regulation of transcription, differentiation, apoptosis, and the cell cycle. Regulation of these functions is mediated by phosphorylation at as many as 16 cyclin-dependent kinase (CDK) phosphorylation sites in vivo. The contribution of these sites to the regulation of the various Rb functions is not well understood. To characterize the effect of phosphorylation at these sites, we systematically mutagenized the serines or threonines to glutamic acid. Thirty-five mutants with different combinations of modified phosphorylation sites were assayed for their ability to arrest the cell cycle and for their potential to induce differentiation. Only the most highly substituted mutants failed to arrest cell cycle progression. However, mutants with as few as four modified phosphorylation sites were unable to promote differentiation. Other mutants had increased activity in this assay. We conclude that modification of Rb phosphorylation sites can increase or decrease protein activity, that different Rb functions can be regulated independently by distinct combinations of sites, and that the effects of modification at any one site are context dependent.