Metoprolol is available for clinical use as a racemic mixture. The S-(-)-metoprolol enantiomer is the one expressing higher activity in the blockade of the β(1) -adrenergic receptor. The α-hydroxymetoprolol metabolite also has activity in the blockade of the β(1) -adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α-hydroxymetoprolol in plasma using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). 1-ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α-hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC-MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α-hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100-mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)(0-∞) values for S-(-)-metoprolol (AUC(S-(-)) /AUC(R-(+))  = 1.81) and the favoring of the formation of the new chiral center 1'R of α-hydroxymetoprolol (AUC(0-∞) (1'R/1'S)  = 2.78). Chirality, 2012. © 2012 Wiley Periodicals, Inc.