Viral polypeptides labeled with [35S]methionine in mouse L fibroblasts infected with either of two strains (JHM or MHV3) of mouse hepatitis virus (MHV) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four major polypeptides of apparent molecular weights, 180,000 (p′180′), 56,000 (p′56′), 24,000 (p′24′), and 22,000 (p′22′) were detected after a 15-min pulse of methionine. During a 2-hr chase performed late in infection (5.5 hr PI) a polypeptide of molecular weight 50,000 (p′50) was observed which appears to be derived from p′56′ by proteolytic processing. Both p′56′ and p′50′ were enriched in the amino acid arginine. Of the five major viral polypeptides only p′180′ could be labeled by growing infected cells in the presence of [14C]glucosamine; moreover, it was the only polypeptide whose mobility of PAGE was altered after labeling infected cells with [35S]methionine in the presence of glycosylation-inhibiting drugs. Despite the extreme similarity in polypeptide patterns specified by JHM and MHV3, the apparent molecular weights of each of the five major JHM polypeptides were consistently lower (by about 500-1000) daltons than the corresponding ones of MHV3. © 1979.