Cell cycle-dependent phosphorylation appears to control the function of the retinoblastoma gene product, p110RB1. We have previously shown that both the N-terminal portion and the portion corresponding to exon 23 are important in the hyperphosphorylation of p110RB1. To gain further insight into the domains involved in these phosphorylations, we have created a series of mutations in the murine cDNA and investigated their effects on phosphorylation, protein conformation and transcription of genes controlled by an E2 element. Three series of p110RB1 constructs were made: (1) mutations and/or deletions in the cluster of potential p34cdc2 consensus sequences of exon 23, (2) deletions for the entire region C-terminal to the large T antigen (TAg)-binding domains or (3) mutations in eight potential p34cdc2 sites, including four in exon 23. Following transfections into COS cells, phosphorylation, TAg binding and protein conformation were evaluated. Constructs were also tested for their ability to repress transcription from the adenovirus early promoter. All mutant proteins bound to SV40 TAg. Mutation of Ser-781, Ser-787 and Ser-788 had no detectable effect on the phosphorylation pattern of p110RB1, producing both the fast-migrating, hypophosphorylated and slower migrating, hyperphosphorylated bands on SDS-PAGE gels. However, mutation or deletion of Ser-800 and Ser-804 resulted in proteins which failed to show the characteristic shift in molecular weight associated with the hyperphosphorylated form of p110RB1. A protein mutated only at Pro-805, the residue adjacent to Ser-804, behaved similarly to mutants with both Ser-800 and Ser-804 altered; mutation of Pro-801 had no effect on the mobility of the hyperphosphorylated species. While differences were seen in the ability of these proteins to be hyperphosphorylated, the wild-type function of repression of adenovirus early promoter activity was retained. These results are consistent with the large shift in apparent molecular weight of p110RB1 upon hyperphosphorylation being due to phosphorylation of Ser-804. Phosphorylation of this p34cdc2 consensus sequence and the subsequent conformational change in p110RB1 detected in denaturing gels is not necessary, however, for the phosphorylation-dependent modulation of p110RB1-TAg interaction.