The metabolic fate of [14C]clenbuterol was studied in male and female Wistar rats. After a single oral dose of 200 microgram/kg [14C]clenbuterol, in an 8-day study period, approximately 60% of the radioactivity was eliminated in urine; 20 and 30% of the radioactivity was excreted in feces by male and female rats, respectively. HPLC coupled to on-line radioactivity detection allowed the separation and quantitation of clenbuterol metabolites, some of which were found to be poorly stable in urine. Most of the urinary and fecal metabolites of clenbuterol were isolated and identified using various MS techniques. Analytical methods were also developed to establish the metabolic profiles in feces and tissues, up to 72 hr after clenbuterol administration. Clenbuterol was mainly metabolized by N-dealkylation (secondary amine), as well as N-oxidation and sulfate conjugation (primary amine). Gender-related differences in the rates of clenbuterol N-dealkylation were observed. 4-N-Hydroxylamine was the major metabolite detected in urine, whereas more than one half of the radioactivity in feces was associated with clenbuterol sulfamate.