The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11-27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.