The synthesis and processing of simian rotavirus SA11 polypeptides was investigated after infection of MA104 cells. [35S]methionine- or 3H-amino acid-labeled cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was maximal 3 to 5 h postinfection, and 12 major viral polypeptides were detected. Immunoprecipitation and peptide mapping experiments, demonstrated five viral structural proteins (125,000 daltons [125K], 94K, 88K, 41K, and 38K). Three proteins (53K, 35K, and 34K) were identified as nonstructural by comparison of their partial proteolysis maps with those from polypeptides of similar molecular weight synthesized in vitro from viral RNA transcripts. Assignment as to structural or nonstructural status of two other primary gene products (26K and 20K) remains tentative. Pulse-chase experiments and tunicamycin blockage of glycosylation revealed cotranslational or post-translational modifications (or both) and precursor-product relationships of several of the polypeptides. Tunicamycin inhibition of glycosylation identified a 35.5K polypeptide which was proven to be the precursor to the 38K structural glycoprotein by immunoprecipitation and peptide mapping analyses. Tunicamycin treatment of infected cells also resulted in the disappearance of other glycoprotein species (23K to 29K) and in the concomitant build-up of an unglycosylated 20K polypeptide, suggesting a precursor-product relationship between those polypeptides. Labeling with [3H]glucosamine or [3H]mannose suggested that the rotavirus glycoproteins contained high mannose oligosaccharides. The effects of amino acid analogs on rotavirus polypeptide synthesis and processing were also investigated.