The Pseudomonas aeruginosa PAK pilus is capable of mediating the binding of this strain to human respiratory epithelial cells. We have produced monoclonal antibodies (MAbs) to the PAK pilus in order to elucidate the location of the binding domain of the pilus for human buccal epithelial cells (BECs). Four MAbs are described. MAbs PK41C and PK34C were found to react with P. aeruginosa pilins produced by a large number of strains. The epitope recognized by PK41C was determined to lie within the N-terminal region of the pilin and is likely constituted by amino acid residues 22 through 33. The epitope for PK34C was located in the C-terminal region of the pilin and was partially dependent on an intact intrachain disulfide bridge between cysteine residues 129 and 142. PK99H and PK3B were found to react specifically with PAK pilin. The epitope for PK99H was also localized in the C-terminal region of the pilin protein and appears to reside between amino acid residues 130 and 138. The epitope for PK3B was not localized by using the methods of this study, but it is likely dependent on the three-dimensional structure of the pilin. Fab fragments of PK99H inhibited adhesion of strains PAK and 492c to BECs, but the adherence of five other strains was not affected. Fab fragments of PK34C inhibited adhesion of all piliated strains examined. Fab fragments from both of these antibodies inhibited PAK pilus binding to BECs. Fab fragments of PK41C and PK3B had no effect on P. aeruginosa binding to BECs. These results confirm that the C-terminal region of the pilin has adhesin qualities and that a conserved epitope lies within this region.