The potential polyspecificity of an antipeptide monoclonal antibody was systematically examined using a soluble synthetic combinatorial library (SCL). This SCL was composed of 400 different hexapeptide mixtures, each of which consisted of more than 130,000 peptides totalling 50 million individual sequences in approximately equimolar concentration. The 400 peptide mixtures making up this SCL were screened by competitive enzyme-linked immunosorbent assay (ELISA) for their ability to inhibit monoclonal antibody binding to the original immunizing peptide. Individual peptides were derived from three different peptide mixtures of the peptide library through an iterative screening and selection process. In addition to the identification of the six-residue antigenic determinant recognized by this monoclonal antibody, two other hexapeptides were found to have binding affinities 5- to 10-fold higher than the original antigenic determinant sequence. These peptide sequences represent analogs in which a polar amino acid (threonine) in the original antigenic determinant was substituted with a large, aromatic amino acid (either tryptophan or tyrosine). Peptide analogs of the antigenic determinant, ranging from single to multiple substitutions, as well as peptide sequences completely unrelated to the immunizing peptide, were also identified having binding affinities comparable to the original immunogen. The present study illustrates the power of SCLs for the determination of alternative binding motifs for antigen antibody interactions. The use of SCLs in this manner may help to elucidate the extent of cross-reactivity, polyspecificity and molecular mimicry found in antigen-antibody interactions.