Background IL-16, a multifunctional cytokine with increased expression in the airways of asthmatic subjects, inhibits allergic airway inflammation in animal models. A T→C single nucleotide polymorphism (SNP) at the −295 position in the promoter region of the IL16 gene has been described. Objective We sought to examine the functional significance of this promoter SNP and its relationship to asthma. Methods We examined the effect of the −295 SNP on promoter activity in cell-line (HBE4-E6/E7) transfection experiments. We investigated the association of the IL16 −295 genotype with asthma among 341 affected sib-pair white families and 184 unrelated nonasthmatic control subjects. We analyzed the association between the IL16 genotype and asthma using family-based association test and case-control analyses. Results In in vitro transfection experiments the T allele in the −295 position was associated with substantially reduced promoter activity compared with the C allele. In the family study the more common T allele at the −295 position was significantly associated with all asthma phenotypes (P = .002 to P = .015). In the case-control analysis asthmatic subjects were more likely than unrelated nonasthmatic control subjects to have the −295 TT genotype, but this did not reach statistical significance (odds ratio, 1.36; 95% CI, 0.92-2.02). Conclusions The T allele at the −295 position in the IL16 promoter region is associated with reduced promoter activity relative to the C allele and with asthma in this white population. Further investigation is needed to delineate the mechanisms underlying these findings and the relationship of the IL16 −295 genotype to asthma in other populations.