The reovirus attachment protein, 1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The 1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of 1 that binds cell surface carbohydrate. Chimeric and truncated 1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-1 antibodies, and oligomerization indicates that the chimeric and truncated 1 proteins are properly folded. To assess carbohydrate binding, recombinant 1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated 1 proteins, the sialic acid-binding domain of type 3 1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted ␤-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of 1 protein purified from virions. In contrast, the homologous region of T1L 1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required.Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 1 tail. Furthermore, our findings indicate that T1L and T3D 1 proteins contain different arrangements of receptor-binding domains.