Like other intronless G-protein coupled receptor genes, the ₂-adrenergic receptor (₂AR) has minimal genetic space for population variability and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies utilize the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5'UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to ₂AR 5'-flanking sequence, 3'-biotin labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies 5% (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance was observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and two increased binding, while five showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only ~50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which ₂AR 5'-flanking polymorphisms affect obstructive lung phenotypes.