Genetic variations in the detoxification enzyme glutathione S-transferase P1 (GSTP1) may modify the teratogenicity of lifestyles, such as smoking. We investigated the role of the I105V polymorphism in GSTP1, parental periconception smoking, and their interaction with nonsyndromic cleft lip with or without cleft palate (CL/P) risk in the offspring. The GSTP1 I105V polymorphisms were determined in Dutch nonconsanguineous Caucasians comprising of 155 CL/P triads (mother, father, child) and 195 control triads. The analyses were also carried out on complete triads only (n = 69 CL/P and n = 95 controls). Transmission disequilibrium testing and logistic regression analyses were performed. Neither maternal nor paternal smoking increased CL/P risk; odds ratios (OR): 1.2, 95 confidence intervals (CI) 0.7 -2.0 and OR: 1.0, 95% CI = 0.6 -1.6, respectively. Carriership of the polymorphic Val105 allele in mothers may increase CL/P risk, OR: 1.5, 95% CI = 0.96 -2.5. Children homozygous for the Val105 allele may show an increased risk of CL/P, OR: 2.2, 95% CI = 0.8-6.4. Maternal smoking tended to increase CL/P risk in mothers and children carrying Val105 alleles, OR = 1.9, 95% CI = 0.9 -4.0 and OR = 2.2, 95% CI = 0.98 -4.9, respectively. The highest risk for CL/P in children carrying Val105 alleles with a smoking father was 1.7, 95% CI = 0.8 -3.5. The GSTP1 I105V polymorphism in mothers and/or children either alone or in combination with maternal smoking may contribute to CL/P risk. Although of borderline significance, these results may underline the importance of smoking cessation in the periconception period for the prevention of CL/P in future generations.