Based on nucleotide sequence analysis of the hemagglutinin (HA) gene from the virulent and avirulent A/chicken/Pennsylvania/83 influenza viruses, it was previously postulated that acquisition of virulence was associated with a point mutation that resulted in loss of a glycosylation site. Since there are two potential glycosylation sites in this region of the HA molecule and since all Asn-Xaa-Thr/Ser sequences in the HAs of different strains are not necessarily glycosylated, the question remained open as to whether either one of these sites was glycosylated. We now provide direct evidence that a site-specific glycosylation affects cleavage of the influenza virus HA and thus virulence. We have identified the glycosylation sites on the HA1 subunit from the virulent and avirulent strains by direct structural analysis of the isolated proteins. Our results show that the only difference in glycosylation between the HA1s of the virulent and avirulent strains is the lack of an asparagine-linked carbohydrate on the virulent HA1 polypeptide at residue 11. Further, we show that the HA1s of both the avirulent and virulent viruses are not glycosylated at one potential site, while all other sites contain carbohydrate. Amino acid sequence analysis of the HA1 of an avirulent revertant of the virulent strain confirmed these findings.