The SELH/Bc mouse strain (SELH) has a high frequency of the lethal neural tube closure defect, exencephaly, in newborns and embryos. Previous work has shown that all SELH mouse embryos have an abnormal mechanism of rostral neural tube closure. They lack initiation of contact and fusion of the cranial neural tube at the prosencephalon/mesencephalon boundary [Closure 2), and undergo closure by extension of a more rostral site of fusion. This process fails in 10-20% of embryos, where the mesencephalic folds remain unelevated, resulting in exencephaly. Previous work has also shown that the cause of liability to exencephaly in SELH mice is multigenic, involving a small number of loci. The purpose of the present study was to test the hypothesis that the genes causing the lack of Closure 2 also cause the liability to exencephaly in SELH, by observation of their joint transmission from genetically segregating animals. A concurrent mapping study provided the necessary genetic material, a segregating F2 generation from a cross of SELH with the normal LM/Bc strain. The genetic liability to exencephaly transmitted by individual F2 sires had been measured by the frequencies of exencephalic day 14 embryos they produced in test-crosses with SELH females. A selected subset of 13 of these test-crossed F2 sires was bred with a second set of SELH females, and the embryos were examined earlier, during the period of neural tube closure, on days 8 and 9 of gestation, to determine the presence of Closure 2. Six F2 sires were among the highest exencephaly producers (6-11%), six were among the lowest (0%), and one was intermediate (5%). Among embryos at the appropriate stage for scoring, the presence of Closure 2 was observed to be inversely correlated with the later risk of exencephaly, being present in 93% (71/76) from the low-risk sires and 35% (36/103) from the high-risk sires. In each case, the remaining embryos had a closure mechanism like that of SELH embryos. Among the individual intermediate- and high-risk sires, there was also a clear correlation between the frequency of exencephaly in older embryos and the frequency of lack of Closure 2 in early embryos (rs = 0.88; P < 0.05). This study demonstrates that high liability to exencephaly and absence of Closure 2 are genetically transmitted together. That is, the cause of the lack of Closure 2 in SELH mice is shown to be also the probable cause of the high liability to exencephaly.