Orofacial clefts have been associated with maternal cigarette smoking and lack of folic acid supplementation (which results in higher plasma homocysteine concentrations). Because endothelial nitric oxide synthase (NOS3) activity influences homocysteine concentration and because smoking compromises NOS3 activity, genetic variation in NOS3 might interact with smoking and folic acid use in clefting risk. The authors genotyped 244 infants with isolated cleft lip with or without cleft palate (CL/P), 99 with isolated cleft palate, and 588 controls from a California population-based case-control study (1987-1989 birth cohort) for two NOS3 polymorphisms: A(-922)G and G894T. Analyses of gene-only effects for each polymorphism revealed a 60% increased risk of CL/P among NOS3 A(-922)G homozygotes (odds ratio (OR) = 1.6, 95% confidence interval (CI): 1.0, 2.6). There was some evidence for higher risk of CL/P with maternal periconceptional smoking in infants with an NOS3 -922G allele (for homozygotes, OR = 2.5, 95% CI: 1.2, 5.6) but not in those with an 894T allele. For CL/P risk, odds ratios were over 4 among mothers who smoked, who did not use vitamins, and whose infants had at least one variant allele for each NOS3 polymorphism (for A(-922)G, OR = 4.6, 95% CI: 2.1, 10.2; for 894T, OR = 4.4, 95% CI: 1.8, 10.7). No similar patterns were observed for risk of cleft palate.