Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin–keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin–horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen. According to their cross-reactivities with the other β-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (IgG1) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG2a) had marked cross-reactivities with most of the 17 tested β-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%). The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml–1 (Mab 1D1). To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 µg ml–1 gel for ampicillin and cloxacillin, respectively. Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.