The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein σ2. The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to σ2. The deduced σ2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions. Predictions of σ2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure. A larger domain is suggested to be formed from the amino-terminal three-fourths of σ2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region. The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an α-helix. A region of striking similarity was found between amino acids 354 and 374 of σ2 and amino acids 1008 and 1031 of the β subunit of the Escherichia coli DNA-dependent RNA polymerase. We suggest that the regions with similar sequence in σ2 and the β subunit form amphipathic α-helices which may play a related role in the function of each protein. We have also performed experiments to further characterize the double-stranded RNA-binding activity of σ2 and found that the capacity to bind double-stranded RNA is a property of the σ2 protein of prototype strains and of the S2 mutant tsC447.