We have examined the functional consequences of mutations present in defective alleles of the retinoblastoma susceptibility gene (RB1) isolated from two spontaneously arising tumors. Unlike cDNA clones expressing the wild-type protein p110Rb, those encoding the two mutant proteins failed to induce the appearance of senescent cells in transfected Saos-2 human osteosarcoma cells. The mutant proteins were also defective in binding to the E1A oncoprotein, were unable to become hyperphosphorylated, and failed to become tightly associated with nuclear structures. We conclude that mutations in two distinct regions of the protein concomitantly affect these four aspects of p110Rb function.