Association of the reovirus proteins σ3 and μ1 influences viral entry, initiation outer capsid assembly, and modulation of the effect of σ3 on cellular translation. In this study, we have addressed whether structural changes occur in σ3 as a result of its interaction with μ1. Using differences in protease sensitivity to detect conformationally distinct forms of σ3, we showed that association of σ3 with μ1 caused a conformational change in σ3 that converted it from a protease-resistant to a protease- sensitive structure and occurred posttranslationally. The effect of μ1 on the structure of σ3 was stoichiometric. Our results are consistent with a model in which σ3's association with μ1 shifts its function from translational control to assembly of an outer capsid in which σ3 is folded into the protease-sensitive conformation that is required for its cleavage during the next round of infection.