For many years, peptic-tryptic digests of gliadin, known as Frazer's fraction III, have been used in investigations of gliadin effects. Potential contamination by the proteases pepsin and trypsin, however, was not considered. To investigate the influence of contaminating proteases on binding of gliadin peptides to rat small intestinal brush border membranes we compared binding characteristics of different gliadin digests. Binding of biotinylated probes was studied in dot blots and Western blots with an enhanced chemiluminescence system. In gliadin peptide preparations only contaminating trypsin, but not pepsin, was detectable by specific antisera. Digestion with insoluble proteases attached to cross-linked beaded agarose yielded gliadin peptides free of contaminating pepsin and trypsin. These peptides bound 30% less to brush border membranes. Using these peptides, there was no trypsin-typical binding pattern to low molecular mass membrane proteins in contrast to peptide preparations which contained contaminating trypsin. In conclusion contaminating trypsin might alter gliadin peptide binding characteristics by direct binding to brush border membranes and by interfering with interactions between gliadin peptides and brush border membranes.