The mouse ephA8 gene is expressed in a rostral-to-caudal gradient in the developing superior colliculus, and these EphA gradients may contribute to the proper development of the retinocollicular projection. Thus, it is of considerable interest to elucidate how the ephA8 gene expression is controlled by upstream regulators during the development of the mesencephalon. In this study, we employed in vivo expression analysis in transgenic mouse embryos to dissect the cis-acting DNA regulatory region, leading to the identification of a CGGTCA sequence critical for the ephA8 enhancer activity. Using this element as the target in a yeast one-hybrid system, we identified a Meis homeobox transcription factor. Significantly, DNA binding sites for Pbx, another TALE homeobox transcription factor, were also identified in the ephA8 enhancer region. Meis2 and Pbx1/2 are specifically expressed in the entire region of the dorsal mesencephalon, where specific colocalization of EphA8 and Meis is restricted to a subset of cells. Meis2 and Pbx2 synergistically bind the ephA8 regulatory sequence in vitro, and this interaction is critical for the transcriptional activation of a reporter construct bearing the ephA8 regulatory region in the presence of histone deacetylase inhibitor. More importantly, when expressed in the embryonic midbrain, the dominant-negative form of Meis down-regulates endogenous ephA8. Interestingly, we found that both Meis2 and Pbx2 are constitutively bound in the ephA8 regulatory region in the dorsal mesencephalon. These studies strongly suggest that Meis and Pbx homeobox transcription factors tightly associate with the ephA8 regulatory sequence and require an additional unidentified regulator to ensure the specific activation of ephA8.