The issue of whether regular use of an inhaled β2-adrenergic agonist worsens airflow and clinical outcomes in asthma is controversial. Retrospective studies have suggested that adverse effects occur in patients with a genetic polymorphism that results in homozygosity for arginine (Arg/Arg), rather than glycine (Gly/Gly), at aminoacid residue 16 of the β2-adrenergic receptor. However, the existence of any genotype-dependent difference has not been tested in a prospective clinical trial. Patients with mild asthma, not using a controller medication, were enrolled in pairs matched for forced expiratory volume in 1 s (FEV1) according to whether they had the Arg/Arg (n=37; four of 41 matches withdrew before randomisation) or Gly/Gly (n=41) genotype. Regularly scheduled treatment with albuterol or placebo was given in a masked, cross-over design, for 16-week periods. During the study, as-needed albuterol use was discontinued and ipratropium bromide was used as needed. Morning peak expiratory flow rate (PEFR) was the primary outcome variable. The primary comparisons were between treatment period for each genotype; the secondary outcome was a treatment by genotype effect. Analyses were by intention to treat. During the run-in period, when albuterol use was kept to a minimum, patients with the Arg/Arg genotype had an increase in morning PEFR of 23 L/min (p=0·0162); the change in patients with the Gly/Gly genotype was not significant (2 L/min; p=0·8399). During randomised treatment, patients with the Gly/Gly genotype had an increase in morning PEFR during treatment with regularly scheduled albuterol compared with placebo (14 L/min 95% CI 3 to 25; p=0·0175). By contrast, patients with the Arg/Arg genotype had lower morning PEFR during treatment with albuterol than during the placebo period, when albuterol use was limited (–10 L/min –19 to –2; p=0·0209). The genotype-attributable treatment difference was therefore –24 L/min (–37 to –12; p=0·0003). There were similar genotype-specific effects in FEV1, symptoms, and use of supplementary reliever medication. Genotype at the 16th aminoacid residue of the β2-adrenergic receptor affects the long-term response to albuterol use. Bronchodilator treatments avoiding albuterol may be appropriate for patients with the Arg/Arg genotype.