Abstract: Polyubiquitination can mediate several different biochemical functions, determined in part by which lysine of ubiquitin is used to link the polyubiquitin chain. Among the HECT domain ubiquitin ligases, some, such as human E6AP, preferentially catalyze formation of K48-linked polyubiquitin chains, while others, including Saccharomyces cerevisiae Rsp5 and human Itch, preferentially catalyze formation of K63-linked chains. The features of HECT E3s that determine their chain type specificities have so far not been identified. We show here that chain type specificity is a function solely of the Rsp5 HECT domain, that the identity of the cooperating E2 protein does not influence chain type specificity, that single chains produced by Rsp5 contain between 12 and 30 ubiquitin moieties, and that the determinants of chain type specificity are located within the last 60 amino acids of the C lobe of the HECT domain. Our results are also consistent with a simple sequential addition mechanism for polyubiquitination by Rsp5, rather than a mechanism involving the formation of either E2- or E3-linked polyubiquitin chain transfers.