A simple, rapid, and sensitive assay for the simultaneous quantification of the (-)- and (+)-propranolol in human and dog plasma is described using a reversed-phase high-performance liquid chromatography (HPLC) system with fluorescence detection. The method involves extraction of propranolol enantiomers from plasma into 1% 1-butanol in n-hexane at basic pH, followed by evaporation of the organic phase and the formation of diastereomeric derivatives with the chiral reagent (-)-menthyl chloroformate. (+)-Flecainide is used as the internal standard. The limiting concentration of each enantiomer that can be detected is 1 ng/mL plasma. In six normal human volunteers, who received a single oral dose of 80 mg of racemic propranolol, the plasma levels of the (-)-enantiomer were always higher than those of the (+)-enantiomer with a mean (-):(+) ratio of 1.38. The half-lives of both the enantiomers were similar (3.5 +/- 0.3 h). In six normal male mongrel dogs given a single intraportal dose of 40 mg of racemic propranolol, the plasma levels of the (-)-enantiomer were always lower than those of the (+)-enantiomer with a mean (-):(+) ratio of 0.48. The half-life of the (-)-enantiomer (73.3 +/- 16.2 min) was shorter than that of the (+)-enantiomer (87.1 +/- 18.1 min).