The synthesis of avian rotavirus (AvRV-1) polypeptides in MA 104 cells was investigated. Extracts of cells labeled with either [35S]methionine or [3H]mannose were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was detected first at 6 hours postinfection. Ten major viral polypeptides were detected at this time. The presence of five viral structural proteins (100K, 90K, 88K, 45K, and 37K) were demonstrated in AvRV-1-infected cells by immunoprecipitation analysis. One structural polypeptide (37K) was identified as a glycoprotein. Two viral polypeptides (30K and 28K) were identified as nonstructural glycoproteins. By tunicamycin inhibition of glycosylation, a 32K polypeptide was identified. This 32K polypeptide later was proven to be the precursor of 37K structural glycoprotein by immunoprecipitation analysis, beta-N-acetylglucosaminidase H (Endo H) treatment, and peptide mapping analysis. Avian rotavirus contained high-mannose oligosaccharide content in the glycoproteins similar to the glycoproteins of mammalian rotaviruses.