Swainsonine, an indolizidine alkaloid, inhibits the alpha-mannosidase that is involved in glycoprotein processing. Thus, in cultured animal cells, this alkaloid causes an increase in the surface content of high mannose glycoproteins and a decrease in the amount of complex type glycoproteins (Elbein, A. D., Solf, R., Dorling, P. R., and Vosbeck, K. (1982) Proc. Natl. Acad. Sci. U. S. A., 78, 7393-7397). In this report, the effect of swainsonine on the synthesis virus hemagglutinins was examined. Primary calf kidney cultures were infected with influenza virus and viral replication was allowed to proceed in the absence or presence of swainsonine. Several hours after the addition of swainsonine, [2-3H]mannose or [6-3H]glucosamine were added to label the hemagglutinins and the mature virus particles were isolated. Virus particles raised in the presence of this alkaloid had the same infectivity and hemagglutination titer as virus particles from control cells. However, when the hemagglutinins were examined on sodium dodecyl sulfate gels, the major hemagglutinin (HA0) and its subunits, HA1 and HA2, from swainsonine-treated cells, migrated faster, indicating that they were of lower molecular weights. The labeled hemagglutinins were digested with pronase and the resulting glycopeptides were chromatographed on Bio-Gel P-4. Both the mannose-labeled and glucosamine-labeled glycopeptides from swainsonine-treated virus migrated more slowly on these columns than those of controls cells, suggesting that they were altered in structure. Furthermore, when the glycopeptides were digested with endoglucosaminidase H, 90% of the glycopeptides from swainsonine-treated cells were susceptible to this enzyme, whereas only 30% of those from control cells were digested. The major oligosaccharide released from inhibited cells by endoglucosaminidase H was digestible with alpha-mannosidase, whereas that of control cells was resistant to this enzyme. However, the control cell glycopeptide was digested by a combination of neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, and alpha-mannosidase. These data show that swainsonine prevents the formation of complex glycoproteins and gives rise to increased amounts of high-mannose glycoproteins.