During early brain development mouse Engrailed2 (En2) is expressed in a broad band across most of the mid-hindbrain region. Evidence from gene expression data, promoter analysis in transgenic mice and mutant phenotype analysis in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical role in regulating En2 mid-hindbrain expression. Previously, we identified two Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficient to directed reporter gene expression to the early mid-hindbrain region and showed that the two Pax2/5/8-binding sites are essential for the mid-hindbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the context of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced with the bacterial neo gene by homologous recombination in mouse embryonic stem cells. After transmitting the mutation into mice, the neo gene was removed by breeding with transgenic mice expressing cre from a CMV promoter. Embryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression indistinguishable from that seen in wild type embryos. Furthermore, the mutants did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expression of the endogenous En2 gene. Interestingly, a comparison of the lacZ RNA and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbrain region by persisting longer than the mRNA. The transgene expression directed by the 1.0 kb enhancer fragment therefore does not mimic the entire broad domain of En2 expression. Taken together, these two studies demonstrate that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression.