Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor kappaB (NF-kappaB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus. RIP3 binds RIP through this unique C-terminal segment to inhibit RIP- and TNF receptor-1-mediated NF-kappaB activation. We have identified a unique homotypic interaction motif at the C terminus of both RIP and RIP3 that is required for their association. Sixty-four amino acids within RIP3 and 88 residues within RIP are sufficient for interaction of the two proteins. This interaction is a prerequisite for RIP3-mediated phosphorylation of RIP and subsequent attenuation of TNF-induced NF-kappaB activation.