Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.