Pili are indispensable in adhesion of encapsulated Neisseria meningitidis (MC) to eukaryotic cells. Intrastrain variability with respect to the degree of adhesion is the result of pilin antigenic variation. We have localized the region responsible for this variability to the 20-amino-acid hypervariable domain of pilin. The replacement of an aspartic acid, located in the hypervariable region of a low-adhesive variant by a lysine restored high adhesiveness. To assess whether hyperadhesiveness conferred by some pilin variants was related to the generation of a new pilus-associated ligand, high- and low-adhesive variants were purified. In a first step, low- and high-adhesive pilins were fused to maltose binding protein (MBP). These hybrid proteins bound epithelial cells with the same affinity. Truncated MBP pilin fusions identified a cell-binding domain within the 77 residues of the N-terminal end of mature pilin. This region of the protein is common to low- and high-adhesive derivatives used in this work, thus eliminating the possibility that high adhesiveness conferred by some pilin variants was because of the generation of a new pilus-associated ligand. Electron-microscopic examination showed that low-adhesive derivatives expressed long and distinct pili and adhered as single cells. In contrast, pili of derivatives expressing high-adhesive pilins, either wild type or mutagenized from the low-adhesive variant, formed large bundles which bound bacteria and caused them to grow as colonies on infected monolayers. These data demonstrate that aggregative pili promote high adhesiveness of encapsulated MC.