This study was designed to determine the structures of several unknown, potentially active, monohydroxylated metabolites of (+/-)-propranolol in rats, dogs, and man. The metabolites were isolated from urine by extraction with ethyl acetate at pH 9.6 after enzymatic hydrolysis. They were then separated as their trimethylsilyl and trifluoroacetyl derivatives and detected by flame-ionization GLC or GC/MS. Structure identification of the metabolites was based on a comparison of their GLC retention times and mass spectra with those of the seven synthetic isomeric hydroxypropranolols (HO-P's). Three metabolites not previously described, 2-HO-P, 5-HO-P, and 7-HO-P, as well as the known 4-HO-P, were identified in the rat. 2-HO-P accounted for about 1% of total monohydroxylated propranolol, 5-HO-P for 7 +/- 2% (mean +/- SE), 7-HO-P for 26 +/- 5%, and 4-HO-P for 66 +/- 5%. The separate administration of (+)- and (-)-propranolol demonstrated stereospecific 7-hydroxylation of (+)-propranolol in the rat. The formation of 5-HO-P was selective for the (-)-isomer, whereas 4-hydroxylation was not stereoselective. Also, the recovery of the dose as the monohydroxylated metabolites in urine was considerably higher after (+)-propranolol, 49 +/- 9%, than after (-)-propranolol, 32 +/- 6%. 4-HO-P was the only hydroxylation product in the dog, whereas in man small quantities of 2-HO-P, 5-HO-P, and 7-HO-P were observed in addition to 4-HO-P.