Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here that an unusual gain-of-function mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. Based on a modeled EGL-1/CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4/CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes.