In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within the core Rb-binding sequence abolished pRb and p107 binding in vitro and immortalization activity in vivo. Substitution of amino acids outside the core Rb-binding sequence reduced pRb and p107 binding in vitro and decreased or abolished immortalization of rat embryo fibroblasts in vivo. Although duplication of the Rb-binding domain within the polyomavirus large T antigen results in a molecule that can bind at least twice as much pRb and p107 in vitro, this mutant displayed an essentially wild-type level of immortalization activity. More importantly, we found that the addition of acidic residues within the casein kinase II consensus phosphorylation region flanking the Rb-binding domain, or the deletion of amino acids 256 to 272, increased the immortalizing activity of the mutant polyomavirus large T antigen. These two mutants displayed a greater than wild-type level of pRb binding in vitro, while in contrast, a decreased affinity for p107 binding in vitro was observed. Together, these results indicate that while pRb binding appears to be an essential event for immortalization, there is no tight correlation between the frequency of immortalization and the absolute level of pRb binding in vitro, indicating that other large T antigen functions are important for cellular immortalization.