Rationale: β2-Agonists are the treatment of choice for exerciseinduced bronchoconstriction (EIB) and act through specific receptors (ADRB2). Arg16Gly polymorphisms have been shown to affect responses to regular use of b2-Agonists. Objectives: To evaluate the influence of the Arg16Gly receptor polymorphism on salmeterol bronchoprotection in EIB and assess predictors of bronchoprotection. Methods: A prospective, genotype-blinded, randomized trial was performed in 26 subjects (12 Arg16Arg and 14 Gly16Gly) with EIB who were not on controller therapy. Subjects were administered salmeterol, 50mgtwice a day for 2 weeks,and underwent an exercise challenge 9 hours after the first and last drug dose. In addition to genotype, FEV1, response to salmeterol, degree of EIB, and exhaled nitric oxide (FENO) at baseline were examined for their association with loss of bronchoprotection (LOB). Measurements and Main Results: The maximumexercise-induced FEV1 fall was 27.9 6 1.4% during the run-in period, 8.1 6 1.2% (70.3 6 4.1% bronchoprotection) after the first salmeterol dose, and 22.86 3.2% (18.9611.5% bronchoprotection) after 2 weeks of salmeterol (P = 0.0001). The Arg16Gly polymorphisms were not associated with the LOB in response to salmeterol. FENO values at baseline were significantly related to the LOB (r = 0.47; P = 0.01). Mean change was a 74 6 13% LOB in subjects with FENO levels greater than 50 ppband a 7616%gain in bronchoprotection in those with FENO levels less than 25 ppb (P = 0.01). Conclusions: The LOB that occurs with chronic long-Acting b2-Agonists use is not affected by ADRB2 Arg16Gly polymorphisms. High FENO was associated with marked LOB. Use of long-Acting b2-Agonists before achieving a reduction in FENO may need to be avoided. Clinical trial registered with www.clinicaltrials.gov (NCT 00595361). Copyright © 2013 by the American Thoracic Society.