Background: Salmeterol (a long acting beta2-agonist) is a chiral molecule. (RR)-salmeterol is responsible for pharmacological effect, but basic knowledge of enantioselective pulmonary pharmacodynamics and pharmacokinetics of salmeterol remains unknown. There are safety concerns with (S)-enantiomers of beta2-agonists, with suggestions that these enantiomers may increase bronchial hyperresponsivneness in asthma patients. Methodology: Horses (n = 12) received racemic (rac-) salmeterol 250 μg via inhalation. Enantioselective UPLC–MS/MS was used to determine (R)- and (S)-salmeterol concentrations in pulmonary epithelial lining fluid (PELF) sampled 2, 5, 10 and 15 min after administration, in central lung (endoscopic bronchial biopsy) and peripheral lung (percutaneous pulmonary biopsy) tissues (at 20 and 25 min respectively), and in plasma samples. Results: Physiologically relevant tissue concentrations were found for both enantiomers, with median levels greater in central than peripheral lung (equivalent to 32 and 5 mM (R)-salmeterol for central and peripheral lung respectively). Levels in PELF decreased around 50% over 15 min and enantioselective distribution was observed in the central lung with levels of (R)-salmeterol around 30% higher than (S)-salmeterol. Conclusion: Salmeterol distribution is enantioselective in the central lung. This suggests duration of action is more likely associated with specific B2ADR localisation effects rather than non-specific physiochemical factors which would not be enantioselective.