r4-specific expression of the EphA2 gene is reminiscent of Hoxb1 and its paralog Hoxa1, raising the possibility that EphA2 expression is regulated by Hox genes. Here, we show that 1) the 0.9-kb enhancer of EphA2 contains multiple binding sites for HOXA1/Pbx1 and HOXB1/Pbx1 proteins, 2) oligonucleotides spanning the binding sites bind to these proteins in vitro, and 3) co-expression of HOXA1 and Pbx1 or HOXB1 and Pbx1 proteins transactivates reporter gene expression from the EphA2-r42B enhancer. These results are consistent with recent genetic studies in which EphA2 expression is substantially decreased in hoxa1/hoxb1 double null mutant mice, although an r4-like territory with unknown identity is present in the mutant (14,15). 3 Taken together, these **data** suggest that expression of EphA2 in r4 is, at least in part, directly regulated by Hoxa1 and Hoxb1.