ABSTRACT Pilus-mediated adhesion is essential in the pathogenesis of Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.