A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B-cell epitopes in two different proteins using a peptide-based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15-mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor-h, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non-covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor-h were added to identify potential linear B-cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non-covalent binding although the peptides were in fact present on the non-covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B-cell epitopes in protein antigens.