Two cell lines producing monoclonal antibodies, Abp4 (IgM) and Abp7 (IgG1) against ampicillin were established. The epitopes and the cross-reactions of the antibodies with various β-lactams were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test Abp4 showed broad cross-reaction to human serum albumin (HSA) conjugates of several β-lactams, 6-aminopenicillanic acid and 7-aminocephalosporanic acid. Abp7 reacted only with ampicillin and cephalexin, which have the same acyl side chain. In ELISA inhibition tests, Abp4 inhibited binding to ampicillin-HSA by benzylpenicilloyl-ε-amino-n-caproic acid, and strongly inhibited the binding by penicillamine. Abp7 strongly inhibited the reaction by aminobenzylpenicilloyl-ε-amino-n-caproic acid, but benzylpenicilloyl-ε-aminon-caproic acid was less effected. These data suggest that Abp4 recognizes the thiazolidine ring and Abp7 recognizes the acyl side chain. Therefore, the thiazolidine ring-epitope acts in broad cross-reaction among β-lactams, and the acyl side chain acts only in the cross-reactivity between penam and cephem, which have a similar acyl side chain.