Site-directed mutagenesis was used to study the biological significance of three N-linked glycans (linked to Asn406, Asn448, and Asn463), situated in the CD4-binding region of gp120. Mutagenesis was carried out in a phage M13 system, and the mutated env genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the level of expression affected the biological phenotype of mutant gp120, we expressed the envelope glycoproteins using either a weak (7.5 K) or a strong (11 K) promoter of vaccinia virus. The expression of mutated env proteins was analyzed after infecting CD4-expressing HeLa cells with the r-vaccinia virus, by monitoring the ability of the infected cells to generate CD4-dependent syncytia. Env gene products lacking all three glycans as well as env gene products lacking different permutations of one or two glycans were analyzed. All mutated gp120 species had the expected electrophoretical mobility as anticipated from elimination of one, two, and three N-linked glycans, respectively. Moreover, all mutant env gene products demonstrated the same capacity to induce formation of syncytia, irrespective of using the weak or strong promoter for expression. These data indicate that the three N-linked glycans studied are dispensable for HIV env gene products to function in CD4-binding and the subsequent fusion step.