OBJECTIVES: Studies suggest that pediatric onset of Crohn's disease (CD) may demonstrate more frequent upper intestinal and colonic location and in male gender, in comparison to adults. Variability in age of onset (AOO) and location of disease have not been adequately explained to date. NOD2/CARD15 is highly expressed in the ileum, while TNF-α expression is distributed throughout the gastrointestinal tract. We hypothesized that polymorphisms that affect TNF-α function may influence variability of disease location and AOO of CD. METHODS: We evaluated two CD cohorts based on AOO (pediatric and adult onset) and 100 ethnically matched healthy controls. Patients were evaluated for AOO, disease location, and genotyped for the presence of polymorphisms in NOD2/CARD15 and in the TNF-α promoter region. RESULTS: Early AOO was associated with male gender, upper intestinal involvement, and a polymorphism in the binding site for NF-κB (TNF-863A polymorphism). NOD2 mutations and TNF-863A polymorphism had equivalent but opposite effects on disease location, with a strong combined effect (p = 0.004 corrected for multiple testing). NOD2/CARD15 was associated with ileal involvement, while presence of TNF-863A was inversely associated with ileal disease (OR = 0.42, p = 0.008) and positively associated with isolated colitis (OR = 2.16, p = 0.008, OR = 2.12, p = 0.03 corrected) and familial disease (p = 0.004). CONCLUSIONS: Pediatric onset of CD in our population was associated with a frequent polymorphism in the binding site for NF-κB in TNF-α promoter but not to defined NOD2/CARD15 disease-associated mutations. This polymorphism is associated with colitis and familial disease. NOD2/CARD15 mutations and the TNF-863C/A polymorphism have equivalent but opposite effects on disease location. These findings may help explain differences in CD phenotype.