Folic acid prevents up to 70% of human neural tube defects (NTD), offering the possibility of effective primary prevention for these crippling birth defects. Recent studies with mouse models of NTD aimed to shed light on two unsolved problems of NTD prevention: 1) the mechanism of action of folic acid, and 2) the development of an alternative therapy to prevent NTD that are unresponsive to folic acid. Using a biochemical screening test in cultured mouse embryos, we identified a defect of folate metabolism in embryos homozygous for the splotch (Sp 2H ) mutation that develop anencephaly and spina bifida. In contrast, there are no demonstrable abnormalities of folate metabolism in two other mouse mutants, curly tail and loop-tail, which also develop NTD. Exogenous folic acid prevents up to 50% of NTD in homozygous splotch embryos. Thymidine is also effective in preventing a proportion of NTD in this mutant, suggesting an inborn error in the provision of folate intermediates for pyrimidine biosynthesis in splotch mice. NTD in mutant curly tail mice do not respond to folic acid but are preventable in up to 70% of cases by exogenous myo-inositol. Embryo culture studies show this effect to be mediated via stimulation of protein kinase C and upregulation of retinoic acid receptor β€ expression in the embryonic hindgut. Inositol therapy may represent a possible means of preventing folate-resistant NTD in humans.