The phosphorylation state of the RB protein was found to change markedly during cell differentiation: most of the RB protein was in a highly phosphorylated 115 kD form in human promyelocytic HL60, erythroleukemic K562 and monocytic U937 cells, but during tumor promoter-induced differentiation, the amount of this form decreased and the amount of an under-phosphorylated 110 kD form of the RB protein increased. Induction of differentiation of HL60 cells by dimethylsulfoxide also induced a similar alteration of the RB protein. However, no alteration in the ratio of the two forms was observed in cell lines such as epidermoid carcinoma A431 and adenocarcinoma MKN-7 that were not induced to differentiate by tumor promoter treatment. The highly phosphorylated form and under-phosphorylated form exhibited different affinity for DNA-cellulose and SV40 large T antigen.