Rationale: Beta-2 agonists are the treatment of choice for exercise-induced bronchoconstriction (EIB) and act through specific receptors (ADRB2). Arg16Gly polymorphisms have been shown to affect responses to regular use of beta-2 agonists. Objective: 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 50 mcg b.i.d. for two 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 FeNO at baseline were examined for their association with loss of bronchoprotection (LOB). Measurements and Main Results: The maximum exercise-induced FEV1 fall was 27.9±1.4% during the run-in period, 8.1±1.2% (70.3±4.1% bronchoprotection) after the first salmeterol dose and 22.8±3.2% (18.9±11.5% bronchoprotection) after two 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±13% LOB in subjects with FeNO levels >50ppb and a 7±16% gain in bronchoprotection in those with FeNO levels <25ppb (p=0.01). Conclusions: The LOB that occurs with chronic LABA use is not affected by ADRB2 Arg16Gly polymorphisms. High FeNO was associated with marked LOB. Use of long-acting beta-2 agonists prior to achieving a reduction in FeNO may need to be avoided. Clinical trial registration information available at www.clinicaltrials.gov (NCT00595361).