The rotavirus genome codes for two glycoproteins: an outer capsid structural glycoprotein (VP7, apparent molecular weight 38,000 (38K)) and a nonstructural glycoprotein (NS28K). The synthesis of these glycoproteins was analyzed in infected cells and in a cell-free system derived from rabbit reticulocyte lysates supplemented with dog pancreatic microsomes. The data showed a 37K product synthesized in the cell-free system is the precursor to the 38K glycoprotein and that the 37K polypeptide contains a cleavable signal sequence (apparent molecular weight 1.5K). The 37K polypeptide was glycosylated in vitro in the presence of microsomal membranes to a polypeptide of 38K that was immunoprecipitated by monospecific antiserum to VP7. Endo H digestion of the 38K polypeptides from either infected cells or the cell-free system produced polypeptides of identical molecular weight, 35.5K (the glycoprotein precursor lacking the signal sequence). These results were confirmed by comparative studies with a variant of SA11 that is defective in glycosylation of VP7. Similar experiments with the 20K precursor to the 29K nonstructural glycoprotein showed the 20K polypeptide contains a noncleavable signal sequence. Both glycoproteins were inserted into microsomal membranes and were processed via oligosaccharide trimming.