The drs gene was originally isolated as a suppressor against v-src transformation. Expression of drs mRNA was markedly downregulated in a variety of human cancer cell lines and tissues, suggesting that the drs gene acts as a tumor suppressor. In this study, we found that ectopic expression of the Drs protein induced apoptosis in human cancer cell lines. Analyses using deletion mutants of drs revealed that both the C-terminal region and the three consensus repeats in the N-terminal region are essential for the induction of apoptosis. Caspase-12, -9, and -3 were sequentially activated by drs, and specific inhibitors of caspase-3 and -9 suppressed drs-induced apoptosis. The release of cytochrome c from the mitochondria into the cytoplasm was not observed in apoptosis by drs, suggesting that the mitochondrial pathway does not mediate drs-induced apoptosis. Furthermore, we found that the Drs protein can interact with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein localized in the endoplasmic reticulum, and that coexpression of these genes increased the efficiency of apoptosis. These results indicated that Drs induces apoptosis by a novel pathway mediated by ASY/Nogo-B/RTN-x(S), caspase-12, -9, and -3.