The incorporation of 35SO 4 2− into viral polypeptides in MDBK cells infected with influenza virus was analyzed by SDS-polyacrylamide gel electrophoresis. When infected cells were labeled in the absence of calf serum, three polypeptides, HA, NP, and M, were resolved in isolated plasma membranes, and HA was the only polypeptide in which 35SO 4 2− incorporation was detected. Sulfation of HA was also demonstrated in both smooth and rough cytoplasmic membranes, whereas there was no detectable 35SO 4 2− incorporation into unglycosylated proteins. These results indicate that at least partial sulfation of HA is already completed at rough membranes. However, when infected cells were doubly labeled with [ 3H]leucine and 35SO 4 2−, the 35 S 3 H ratio in the HA polypeptide was not uniform in virions and subcellular components. The ratio was highest in virions, and decreased in the order of virions, plasma membranes, smooth membranes, and rough membranes, suggesting that further sulfate incorporation may occur in smooth membranes and plasma membranes as well as in rough membranes. The 355/3H ratio of HA associated with plasma membranes varied with the length of labeling; higher ratios were observed with shorter labeling periods. This observation may be explained by sulfate incorporation into performed HA. Significant amounts of 35SO 4 2− incorporation into HA were found in the presence of cycloheximide, at concentrations wich completely inhibited the synthesis of viral polypeptides. Further, pulse-labeling of infected cells with 35SO 4 2− at various times after inhibition of protein sy thesis by cycloheximide showed that sulfation of HA polypeptides continues to occur as long as 30 min or more after synthesis, which also suggests that 35SO 4 2− continues to be incorporated into HA polypeptides even after they migrate from rough membranes. The acceptors for sulfation appear to be oligosaccharide units of viral glycoproteins since almost all 35S label was recovered in association with glycopeptides after exhaustive digestion of virions with Pronase followed by gel filtration. As was observed for HA, the incorporation of 35SO 4 2− into cellular mucopolysaccharides was also observed in every subcellular fraction tested. Further, when either smooth or rough cytoplasmic membranes isolated from infected cells were incubated with 3′-phosphoadenosine-5′-phosphosulfate ([ 35S]PAP) in vitro, sulfate incorporation into mucopolysaccharide was detected, which suggests that sulfation of mucopolysaccharide occurs in both smooth and rough membranes in vivo. Additionally, it was found that the rate of incorporation of 35SO 4 2− into cellular mucopolysaccharide was markedly inhibited by influenza virus infection.