In the hindbrain of the mouse embryo, there is often coincident rhombomere-restricted expression of Eph receptor tyrosine kinases and Hox homeobox genes, raising the possibility of regulatory interactions. In this paper, we have identified cis-acting regulatory sequences of the EphA2 (Eck) gene, which direct node and hindbrain-specific expression in transgenic embryos. An 8-kilobase region of mouse genomic DNA element was sufficient to drive rhombomere 4 (r4)-specific expression while conferring patchy expression in the node. Further analysis localized the rhombomere-specific enhancer to a 0.9-kilobase sequence. This element contains multiple Hox-Pbx consensus binding sites that bind to both HOXA1/Pbx1 and HOXB1/Pbx1 proteins in vitro. Co-expression of either HOXA1 or HOXB1 with Pbx1 transactivated EphA2 enhancer-dependent reporter gene expression. These results, together with observations of reduced EphA2 expression in hoxa1 and hoxb1 double mutant mice, suggest that expression of EphA2 gene in rhombomere 4 is directly regulated by Hoxa1 and Hoxb1 homeobox transcription factors.