The genes encoding the glycosyltransferases responsible for the addition of the five sugars in the α-oligosaccharide (α-OS) moiety of lipooligosaccharide (LOS) have been identified. Disruption of these glycosyltransferase genes singly or in combination results in corresponding truncations in LOS. In the present work we show that sequential deletion of the terminal four sugar residues of gonococcal α-OS had no discernible effect on the invasion of human conjunctival, endometrial, and cervical cell lines. However, deletion of the proximal glucose, which resulted in the complete deletion of α-OS, significantly impaired invasion of the gonococci into all three cell lines. The effect of deleting α-OS on invasion was independent of and additive to the known invasion-promoting factor OpaA. These data suggest that the proximal glucose residue of the α-OS chain of LOS is required for efficient invasion of gonococci into host mucosa.