Antigen recognition sites of antibodies to haptens were investigated by use of mAbs (MA-7 and -15) against MA. The affinity constants of MA-15 and its heavy and Light chains for MA were estimated by ELISA, to be 3 x 10(8)/M, 2 x 10(7) and 2 x 10(6)/M, respectively. Messenger RNA purified from mAb-secreting hybridoma was amplified by RT-PCR and its nucleotide and amino acid sequences of the mAb was determined. Each CDR peptide of the heavy chain of the mAb was synthesized and the affinity constant of the mAb was estimated by competitive ELISA. The affinity constant of CDRH-2 of MA-7 was assumed to be 2 x 10(6)/M. The magnitude of this value almost corresponds to that of the association constant of many enzymes. Based on the spatial conformations of the mAbs, CDRH-3 is not considered to participate in the antigen recognition. MA-7 reacts with morphine, as expected from the high level of homology of the amino acid sequence of CDRH-2. These findings suggest that CDRH-2 plays a major role in the recognition of MA. Based on the results of a comparison of the molecular structures of MA analogues, anti-MA mAb may recognize phenylalanine and the N-R skeleton of the antigen.