In previous experiments, we have shown that [I-125]pili of Pseudomonas aeruginosa exhibited specific binding to a low-molecular-weight mucin (MG2) of human submandibular-sublingual saliva (HSMSL; Reddy MS, Levine MJ, Paranchych W, Grit Rev Oral Biol Med 4:315-323, 1993). In the present study, I have utilized unlabeled pill and immunostaining to identify the receptor molecules in HSMSL. In addition to MG2, pill also bound to neutral cystatin (CsnSN), Binding of unlabeled pill to MG2 and CsnSN could be abolished by treatment of HSMSL with trypsin to hydrolyze the peptide moieties or N-acetylation to neutralize the positive charges of the lysine residues. Reductive methylation of HSMSL, which modifies the lysine residues to methyl lysines while retaining the positive charges, did not affect the binding of pill to either MG2 or CsnSN. Further, pill also exhibited binding to a recombinant MG2 peptide (aa 1-86). Collectively, the data suggested that a protein-to-protein interaction via electrostatic forces mediates the binding of the pill to MG2 and CsnSN, Iodination of pill, which modifies tyrosine-24 and/or -27 residues to O-iodotyrosine(s), abolished its binding to CsnSN but not to MG2. These results suggested that the conformation of pill also plays a role in interaction with CsnSN. Conformational change(s) of pill induced by iodination also made it susceptible to hydrolysis with trypsin.