In murine fibroblasts, efficient proteolysis of reovirus outer capsid protein σ3 during cell entry by virions requires the acid-dependent lysosomal cysteine protease cathepsin L. The importance of cathepsin L for infection of other cell types is unknown. Here we report that the acid-independent lysosomal cysteine protease cathepsin S mediates outer capsid processing in macrophage-like P388D cells. P388D cells supported infection by virions of strain Lang, but not strain c43. Genetic studies revealed that this difference is determined by S4, the viral gene segment that encodes σ3. c43-derived subvirion particles that lack σ3 replicated normally in P388D cells, suggesting that the difference in infectivity of Lang and c43 virions is at the level of σ3 processing. Infection of P388D cells with Lang virions was inhibited by the broad spectrum cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane but not by NH 4Cl, which raises the endocytic pH and thereby inhibits acid-dependent proteases such as cathepsins L and B. Outer capsid processing and infection of P388D cells with Lang virions were also inhibited by a cathepsin S-specific inhibitor. Furthermore, in the presence of NH 4Cl, cell lines engineered to express cathepsin S supported infection by Lang, but not c43, virions. Our results thus indicate that differences in susceptibility to cathepsin S-mediated σ3 processing are responsible for strain differences in reovirus infection of macrophage-like P388D cells and other cathepsin S-expressing cells. Additionally, our data suggest that the acid dependence of reovirus infections of most other cell types may reflect the low pH requirement for the activities of most other lysosomal proteases rather, than some other acid-dependent aspect of cell entry.