While studies have demonstrated substantial differences in beta₂-adrenergic agonist enantiomer pharmacology, enantioselective disposition of long-acting beta₂-adrenergic ligand racemic (rac)-formoterol in blood is unexplored after inhaled therapy given analytical challenges. Furthermore, information on enantioselective disposition and partitioning of beta₂-adrenergic agonist in skeletal muscle is absent despite its promising data on muscle anabolism in humans. Using a sensitive UPLC-MS/MS (ultra-high performance liquid chromatography-mass spectrometry) assay, we determined disposition of (R,R)-formoterol and (S,S)-formoterol in plasma and skeletal muscle samples from 11 non-asthmatic men who had inhaled rac-formoterol at therapeutic doses (2×27 μg). Mean (SD) concentrations of (R,R)- and (S,S)-formoterol in plasma and in muscle biopsies of the vastus lateralis 1 h after inhalation of formoterol were 31 (15) and 45 (18) pg×mL^-1 for (R,R)-formoterol and (S,S)-formoterol, respectively, in plasma, and 0.56 (0.32) and 0.51 (0.29) pg×mg_{wet wt}^-1 , respectively, in muscle. Formoterol exhibited different enantioselective disposition in plasma and muscle (p<0.0001). In plasma, mean log (R,R):(S,S)-formoterol ratio was lower than 0 [-0.17(0.07), p<0.0001], whereas in muscle, mean log (R,R):(S,S)-formoterol ratio was slightly higher than 0 [0.04(0.07), p<0.05]. Log (R,R):(S,S)-formoterol ratio in muscle was related to muscle fibre-type composition. Furthermore, formoterol induced an approximately two-fold increase in muscle p-PKA^Ser/thr phosphorylation (p<0.01), indicating a substantial beta₂-adrenergic response. Collectively, these findings suggest that formoterol exhibits modest enantioselective disposition in plasma after inhaled therapy in humans, which appear related to a greater (R,R)-enantiomer disposition in skeletal muscle that may be dependent on fibre-type composition.