Genetic polymorphisms are thought to play an important role in determining susceptibility to neural tube defects (NTDs), for example between different ethnic groups, but the embryonic manifestation of these polymorphic genetic influences is unclear. We have used a mouse model to test experimentally whether polymorphic variations in the pattern of cranial neural tube closure can influence susceptibility to NTDs. The site at which cranial neural tube closure begins (so-called closure 2) is polymorphic between inbred mice. Strains with a caudal location of closure 2 (e.g. DBA/2) are relatively resistant to NTDs, whereas strains with a rostrally positioned closure 2 (e.g. NZW) exhibit increased susceptibility to NTDs. We tested experimentally whether altering the position of closure 2 can affect susceptibility to cranial NTDs, by back- crossing the splotch ( Sp (2H) ) mutant gene onto the DBA/2 background. As a control, Sp (2H) was transferred onto the NZW background, which resembles splotch mice in its closure pattern. Approximately 80% of Sp (2H) homozygotes develop NTDs, both cranial (exencephaly) and spinal (spina bifida). After transfer to the DBA/2 background, the frequency of cranial NTDs was reduced significantly in Sp (2H) homozygotes, confirming a protective effect of caudal closure 2. In contrast, Sp (2H) homozygotes on the NZW background had a persistently high frequency of cranial NTDs. The frequency of spina bifida was not altered in either backcross, emphasizing the specificity of this genetic effect for cranial neurulation. These findings demonstrate that variation in the pattern of cranial neural tube closure is a genetically determined factor influencing susceptibility to cranial NTDs.