Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins σ3, μ1c, and λ2 (core spike) and the cell attachment protein σ1. We focused on σ1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on μ1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D σ3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D σ3 proteins accounted for differences in σ3 epitope maps. Four of 12 non-σ1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different σ1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between σ3/μ1c and σ1 included (i) inhibition by σ3 and μ1c MAbs of σ1-mediated HA, (ii) enhancement of σ1-mediated HA by proteolytic cleavage of σ3 and μ1c, and (iii) genetic studies demonstrating that σ1 controlled the capacity of σ3 MAbs to inhibit HA. These data suggest that (i) epitopes on σ3 and μ1c lie in close proximity to σ1 and that MAbs to these epitopes can modulate σ1-mediated functions, (ii) these spatial relatonships have functional significance, since removal of σ3 and/or cleavage of μ1c to δ can enhance σ1 function, (iii) in nature, the σ1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.