We have investigated whether granzyme B (GzmB) is required for effective cytotoxic T lymphocyte (CTL) mediated control of lymphocytic choriomeningitis virus (LCMV) infection. Clearance of LCMV from tissues of GzmB-deficient (GzmB-) mice following intraperitoneal infection with LCMV was impaired compared with control mice; however, the virus was ultimately eliminated. The impaired clearance of LCMV in GzmB- mice was not due to a deficiency in the generation of LCMV-specific T cells. In addition, CTL from LCMV-infected GzmB- mice efficiently lysed virus-infected cells in vitro, but were deficient in their ability to induce rapid DNA fragmentation in target cells. We examined whether the development of protective immunity against intracranial (i.c.) rechallenge with LCMV was compromised in GzmB- mice. We found that clearance of LCMV from the brain following secondary i.c. infection also was slower in the absence of GzmB; however, the virus was ultimately eliminated and the mice survived. Our data indicate that clearance of LCMV is delayed in the absence of GzmB expression, but that other CTL effector molecules can compensate for the absence of this granule constituent in vivo.