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The morphogenetic process of cranial neural tube closure was studied in normal and mutant splotch mouse embryos to identify factors contributing to the development of the neural tube defect (NTD), exencephaly. The study noted sex differences in the timing of the neurulation process and strain differences in the initiation of cranial neural tube closure, at the site termed closure 2. Splotch embryos exhibit closure 2 at a rostral level, within the forebrain region, and exencephaly results from failure of elevation of the midbrain neural folds in a proportion of homozygotes. Backcrossing the splotch mutation onto the DBA/2 background caused a caudal shift in the position of closure 2, to the midbrain region, and a reduction in the incidence of exencephaly, suggesting that altering the position of closure 2 may aid midbrain fold elevation and prevent the development of exencephaly. Whole-mount in situ hybridisation revealed that expression patterns of Pax2, Pax5 and Fgf8 remained constant within the forebrain- midbrain region, regardless of variation in the position of closure 2 in different strains. NTD in humans can be prevented by periconceptional supplementation with folic acid, although the mechanism of action of folate is unknown. The deoxyuridine (dU) suppression test was adapted for use in whole embryo culture and several genetic models of NTD were screened for defects of folate metabolism. The splotch mutant showed an abnormal dU suppression test, with excessive incorporation of 3H-thymidine. Administration of thymidine or folic acid prevented the NTD in splotch homozygotes, both in vitro and in vivo. Folic acid also ameliorated the excessive incorporation of 3H-thymidine and prevented the neural crest defects in homozygous embryos. The splotch mutant therefore appears to provide a model for folate-preventable NTD in humans.