We report the generation of an anti-idiotypic antibody, clone 12E12, against antisulfamethazine (SMZ) as a surrogate ligand for SMZ in immunoassay procedures. This has been achieved by using the primary monoclonal anti-SMZ antibody as an immunogen in hybridoma technology followed by screening of the resulting hybridromas for binding to the SMZ site of the primary anti-SMZ antibody. Characterization of a strong anti-idiotypic antibody of the betatype, clone 12E12, capable of mimicking SMZ, permitted the development of immunoassay formats for SMZ based on the idiotypic and anti-idiotypic approach and the direct measurement of SMZ content in diluted pig urine. The availability of the strong betatypic anti-idiotypic antibody acting as a surrogate ligand for SMZ enables antibodies to be labeled instead of ligands and offers interesting possibilities for the development of competitive type non-isotopic immunoassays for SMZ.