BACKGROUND: Isolated clubfoot is a common orthopedic birth defect that affects approximately 135,000 newborns worldwide. It is characterized by ankle equinus, hindfoot varus, and forefoot adductus. Although numerous studies suggest a multifactorial etiology, the specific genetic and environmental components have yet to be delineated. Maternal smoking during pregnancy is the only common environmental factor consistently shown to increase the risk for clubfoot. Moreover, a positive family history of clubfoot, in conjunction with maternal smoking, increases the risk 20-fold. These findings suggest that genetic variation in smoking metabolism (xenobiotic) genes may increase susceptibility to clubfoot. Based on this reasoning, we interrogated eight candidate genes from the xenobiotic metabolism. METHODS: Twenty-two single-nucleotide polymorphisms and two null alleles in these genes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, EPHX1, NAT2, GSTM1, and GSTT1) were genotyped in a dataset composed of non-Hispanic white and Hispanic multiplex and simplex families. RESULTS: Only rs1048943/CYP1A1 had significantly altered transmission in the aggregate and multiplex non-Hispanic white datasets (p 5 0.003 and p 5 0.009, respectively). Perturbation of CYP1A1 can cause an increase in harmful, adduct-forming metabolic intermediates. A significant interaction between EPHX1 and NAT2 was also found (p 5 0.007). Importantly, for CYP1A2, significant maternal (p 5 0.03; relative risk [RR] 5 1.24; 95% confidence interval [CI], 1.04-1.44) and fetal (p 5 0.01; RR 5 1.33; 95% CI, 1.13-1.54) genotypic effects were identified, suggesting that both maternal and fetal genotypes can negatively impact limb development. No association was found between maternal smoking status and variation in xenobiotic metabolism genes. CONCLUSION: Together, these results suggest that xenobiotic metabolism genes are unlikely to play a major role in clubfoot; however, perturbation of this pathway may still play a contributory role.