The metabolism of clenbuterol by liver microsomal fractions and precision-cut liver slices was studied in rats and cattle using a 14C-labeled molecule and radio-HPLC quantitation of the resulting metabolites. 4-N-Oxidation of clenbuterol was found to be an extensive in vitro metabolic pathway in both species. Clenbuterol hydroxylamine was by far the major metabolite characterized from microsomal and slice incubation media. Trace amounts of 4-nitro-clenbuterol were also detected. Another important microsomal biotransformation of clenbuterol, resulting in the production of 4-amino-3,5-dichlorobenzoic acid, was observed only when the drug was incubated with bovine liver microsomes. The corresponding glycine conjugate, namely 4-amino-3,5-dichlorohippuric acid, was detected when clenbuterol was incubated with bovine or rat liver slices. Structural characterization of the major metabolites was performed using electrospray ionization-mass spectrometry, either coupled to liquid chromatography or with direct infusion of collected samples. In addition to these compounds, only quantitatively minor metabolites were detected in bovine (but not rat) microsomal incubation media. Analysis of incubation media from liver slices also allowed the quantitation of a few additional metabolites, some of which were shown to be conjugated compounds.