Esmolol is an ultra-short-acting beta-blocker currently in Phase II clinical trials. The ester functionality in esmolol results in rapid metabolism of the beta-blocker into an acidic metabolite and methanol. Dichloromethane was used to denature blood esterases and quantitatively extract esmolol from the blood. A deuterated analogue of esmolol was selected as the internal standard, and both compounds were chromatographed as the trimethylsilyl derivatives. Blood levels of esmolol were quantitated by gas chromatography-mass spectrometry with selective-ion monitoring, focusing on specific ions corresponding to esmolol and the internal standard. The lower limit of sensitivity of the assay was 2.5 ng/mL. Using the assay, blood samples from a dose-ranging study in humans were analyzed for concentrations of esmolol. Steady-state blood levels of esmolol after intravenous infusion rates of 40, 100, 200, 300, 450, and 650 micrograms/kg/min were 0.202, 0.464, 0.977, 1.31, 1.92, and 2.97 micrograms/mL of blood. The elimination t1/2 and total body clearance were estimated to be approximately 10 min and 220 mL/kg/min, respectively. The high clearance of esmolol suggested that metabolism by blood esterase(s) was the primary determinant of the duration of action of the drug.