A quantitative assay for urinary propranolol and six of its unconjugated metabolites by a gas chromatography mass spectrometry computer system has been developed. Conjugated metabolites are also determined following enzyme hydrolysis. These compounds are separated from murine by Amberlite XAD-2 resin column chromatography and are converted to methyl esters and trifluoroacetyl derivatives. Each substance is quantitated from reconstructed ion chromatograms using a specific mass or sum of masses for each compound by comparing its area with that of an appropriate internal standard. The minimum detectable concentrations are in the range of 50--100 ng ml-1. The calibrations are linear from 0.10--15.0 microgram ml-1 with recoveries ranging from 62--91%. This procedure yields a high degree of specificity, selectivity and reproducibility, as demonstrated by the analyses of urine from dogs receiving single 40 mg doses of propranolol orally.