Clenbuterol is a β-adrenergic agonist widely but illegally used in cattle as a growth promoter. The metabolic fate of this drug remains unknown in the main target species, i.e. the bovine, and only limited data have been published concerning its biotransformations in laboratory animals. A metabolic study has been carried out in the rat using ³ H- and ¹⁴ C-labeled clenbuterol. Urine appeared to be the major excretion pathway. Using a soft technique for urine preparation, extraction, and purification, as well as adequate analytical tools in order to preserve labile metabolites, N -oxidation products of the parental drug on the primary amine function were identified for the first time. Clenbuterol hydroxylamine was the major compound, but 4-nitroclenbuterol was also detected. The metabolic pathway leading to the formation of clenbuterol hydroxylamine prevails at high dosages. Clenbuterol hydroxylamine (but not 4-nitroclenbuterol) was also formed extensively when the drug was incubated with rat liver microsomal fractions in aerobic conditions. It is concluded that oxido reduction reactions during urine preparation have previously impaired the identification of this toxicologically important clenbuterol metabolic route.