The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein.