Apoptosis plays an important role in the pathogenesis of reovirus encephalitis and myocarditis in infected animals. Differences in apoptosis efficiency displayed by reovirus strains are linked to the viral mu1-encoding M2 gene segment. Studies using pharmacologic inhibitors of reovirus replication demonstrate that apoptosis induction by reovirus requires viral disassembly in cellular endosomes but not RNA synthesis. Since the mu1 protein functions to pierce endosomal membranes during this temporal window, these findings point to an important role for mu1 in activating signaling pathways that lead to apoptosis. To understand mechanisms used by mu1 to induce apoptosis, a panel of mu1 mutant viruses generated by reverse genetics was analyzed for the capacities to penetrate host cell membranes, activate proapoptotic signaling pathways, evoke cell death, and produce encephalitis in newborn mice. We found that single amino acid changes within the delta region of mu1 reduce the efficiency of membrane penetration. These mutations also diminish the capacities of reovirus to activate proapoptotic transcription factors NF-kappaB and IRF-3 and elicit apoptosis. Additionally, we observed that following intracranial inoculation, an apoptosis-deficient mu1 mutant is less virulent in newborn mice in comparison to the wild-type virus. These results indicate a critical function for the membrane penetration activity of mu1 in evoking prodeath signaling pathways that regulate reovirus pathogenesis.