The metabolism of the anticholinergic drug, aprophen, was studied in rats. In a previous study, the principal metabolite of aprophen (desethyl aprophen) found in the blood of rats which had received an intravenous dose of the drug, was shown to have antimuscarinic activity. Several other metabolites were also found to be present in the urine, as observed by high-performance liquid chromatography (HPLC). In the present study, aprophen was orally administered by intubation into male Sprague-Dawley rats. The animals were then maintained in glass-chambered metabolic cages with food and water ad limitum. The animals were housed for 72 hours, during which time tot;,- urine samples were collected on a 24-hour schedule. Aprophen metabolites in the collected urine were separated and isolated by HPLC and identified by mass spectrometric methods. One major metabolite which was isolated by HPLC and identified as beta-hydroxylaprophen was subsequently synthesized in the laboratory and spectroscopically characterized by mass spectrometric and nuclear magnetic resonance methodologies. The hydroxylated metabolite was shown to have antimuscarinic activity by measuring its inhibition of acetylcholine-induced contraction of guinea pig ileum, of carbachol-stimulated release of alpha-amylase from rat pancreatic acinar cells, and of tritiated N-methylscopolamine binding to rat cortex.