The membrane-spanning domain (MSD) of HIV-1 envelope protein (Env) has an additional glycine residue within a well-conserved putative transmembrane helix-helix interaction motif, GXXXG, and forms a G(690)G(691)XXG(694) sequence (G, glycine; X, any residues; the numbering indicates the position within the Env of an infectious molecular clone, HXB2). Different from vesicular stomatitis virus G (VSV-G), the glycine residues of the GXXXG motif of HIV-1 showed higher tolerance against mutations, and a simultaneous substitution of G690 and G694 with leucine residues only modestly decreased fusion activity and replication capacity of HIV-1. When G691 was further substituted with alanine, phenylalanine or leucine residue while G690 and G694 were substituted with leucine residues, the efficiency of membrane fusion decreased, with the decrease greatest occurring with the leucine substitution, a less severe decrease with phenylalanine, and the least severe decrease with alanine. Substitution with leucine residue also decreased the incorporation of Env onto virions, and the mutant showed the most delayed replication profile. Thus the presence of the extra glycine residue, G691, may increase the tolerance of the other two glycine residues against mutations than VSV-G. The fact that a more severe defect was observed for the leucine residue than the phenylalanine residue suggested that the function of Env depended on the steric nature rather than on the simple volume of the side chain of the amino acid residue at position 691. Based on this result, we propose a hypothetical model of the association among MSDs of gp41, in which G(691) locates itself near the helix-helix interface.