The hemoglobins of the midge Chironomus thummi thummi (Chi t I) are known to cause immediate-type hypersensitivity reactions in humans. Further knowledge of the antigenic sites of such allergens will provide new therapeutic approaches. The aim of our study was to identify and characterize linear B-cell epitopes of the hemoglobin component III of Chi t I (136 amino acid residues). Using the antigenic index algorithm of Jameson and Wolf (Jameson and Wolf (1988) Comput. Appl. Biosci. 4, 181-186), three linear binding sequences of this allergen molecule were predicted. Two mouse monoclonal antibodies (mAbs 3 and 6) raised against purified Chi t I component III were investigated by ELISA for their binding to nine synthetic peptides 19-21 residues in length, covering nearly the whole sequence of component III. MAb 6 recognized only one peptide (11-30) while mAb 3 bound to both N-terminal peptides (1-19 and 11-30), suggesting that the antibody binding site is located in the overlapping region. This assumption could be confirmed in ELISA with solid phase-bound recombinant peptides (RP) as well as in inhibition studies with free tryptic peptides indicating that identification of these linear B-cell epitopes is neither influenced by the method of peptide production nor by the kind of used immunoassay. To define the essential amino acid residues we investigated mAbs with solid phase-bound overlapping octamers. In the case of mAb 3, amino acids experimentally identified as essential for antibody binding (aa 13-17) are identical with those residues predicted as a B-cell epitope with the antigenic index of Jameson and Wolf.