Recent genetic studies have implicated integrins in asthma and atopy susceptibility. We therefore evaluated the integrin-β3 gene (ITGB3), an integrin gene within an asthma linkage peak on chromosome 17, as a candidate for susceptibility to asthma- and atopy-related phenotypes. We genotyped and performed association tests on 19 single nucleotide polymorphisms in ITGB3 in the Hutterites, a founder population, and in three outbred replication populations. Variation in ITGB3 was strongly associated with susceptibility to bronchial hyperresponsiveness and protection from allergic sensitization to mold allergens in this population. Three independent case-control populations representing Caucasians and African Americans were used to replicate this finding, also revealing ITGB3 alleles that are associated with asthma susceptibility and protection from mold allergen sensitization. This study provides evidence that ITGB3 plays a role in the pathogenesis of asthma and sensitization to mold allergens.