The retinoblastoma protein, pRb, plays a pivotal role in G1 to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and C-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the N-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb^CDK (a dominantly active repressive allele of Rb), we have previously shown that restoration of threonine-373 is sufficient to render Rb^CDK sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the N-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we expand this analysis to include additional residues in the N-terminal region of pRb and we further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further found that removal of the C-terminal domain of either RbCDK^+T373 or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Taken together, our data demonstrate a novel function for the N-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.