Intrauterine smoke exposure (IUS) is a strong risk factor for development of airways responsiveness and asthma in childhood. Runt-related transcription factors (RUNX1-3) have critical roles in immune system development and function. We hypothesized that genetic variations in RUNX1 would be associated with airway responsiveness in asthmatic children and that this association would be modified by IUS. Family-based association testing analysis in the Childhood Asthma Management Program genome-wide genotype data showed that 17 of 100 RUNX1 single-nucleotide polymorphisms (SNPs) were significantly (P < 0.03-0.04) associated with methacholine responsiveness. The association between methacholine responsiveness and one of the SNPs was significantly modified by a history of IUS exposure. Quantitative PCR analysis of immature human lung tissue with and without IUS suggested that IUS increased RUNX1 expression at the pseudoglandular stage of lung development. We examined these associations by subjecting murine neonatal lung tissue with and without IUS to quantitative PCR (N = 4-14 per group). Our murine model showed that IUS decreased RUNX expression at postnatal days (P)3 and P5 (P < 0.05). We conclude that 1) SNPs in RUNX1 are associated with airway responsiveness in asthmatic children and these associations are modified by IUS exposure, 2) IUS tended to increase the expression of RUNX1 in early human development, and 3) a murine IUS model showed that the effects of developmental cigarette smoke exposure persisted for at least 2 wk after birth. We speculate that IUS exposure-altered expression of RUNX transcription factors increases the risk of asthma in children with IUS exposure.