Transport of folates in mammalian cells occurs by a carrier-mediated mechanism. The human folate carrier (RFC-1) gene has been isolated and characterized. Within this gene, a common polymorphism, 80A-->G, changing a histidine to an arginine in exon 2 (H27R), was recently identified. Defects in folate metabolism, such as defective carrier molecules, could be implicated in the etiology of neural tube defects (NTDs). In the present case-control study, we recruited 174 Italian probands with nonsyndromic NTD, 43 mothers, 53 fathers and 156 control individuals and evaluated the impact of RFC-1 variant on NTD risk. A statistically significant risk was calculated for the 80GG genotype of the NTD cases (OR=2.35; 95% CI 1.21-4.58) and mothers (OR=2.74; 95% CI 0.92-8.38). On the contrary, the heterozygous genotype of the mothers and both heterozygous and homozygous genotypes of the fathers did not seem to be significant NTD risk factors. Furthemore, according to the multifactorial inheritance of NTDs, we demonstrated that the combined genotypes for MTHFR 1298A-->C and RFC-1 80A-->G polymorphisms of cases resulted in greater NTD risk than heterozygosity or homozygosity for RFC-1 80A-->G variant alone. Conversely, our data provide no evidence for an association between NTD phenotype and combined MTHFR C677T/RFC-1 A80G genotypes. Moreover, here we describe the combinations of the two MTHFR polymorphic sites (677CT and 1298AC) with RFC-1 genotypes. We found that both patients and controls could have at most quadruple-mutation combinations. Interestingly, 27% (7/26) of the mothers and 18.75% (30/160) of the cases genotyped presented four mutant alleles in comparison with 8.5% (11/129) of the controls. Finally, the frequency of NTD cases and mothers carrying combined heterozygosity for the two MTHFR polymorphisms and RFC-1 80GG homozygosity (677CT/1298AC/80GG) (cases=11.3%; mothers 11.5%) was increased compared with controls (1.6%). Altogether, our findings support the hypothesis that RFC-1 A80G variant may contribute to NTD susceptibility in the Italian population.