Zilpaterol is an beta-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLH resulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigen for ELISA development. The average IC(50) derived from the developed zilpaterol immunoassay was 3.94 +/- 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists. The assay was tolerant of up to 10% (v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrations ranging from 0.05 to 1.0 M minimally affected B(0) or IC(50) values. When buffer pH was <7 or >8.8, the IC(50) values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specific zilpaterol ELISA has been developed that can serve as a rapid screening assay.