Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7-117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle, and residues from helices alpha2 and alpha4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb.