Hox genes control cell fates and specify regional identities during vertebrate development. Hox proteins relaxed DNA-binding selectivity in vitro, suggests that Hox specificity of action is achieved in vivo through the action of transcriptional cofactors. Members of the EXD/PBX family of homeoproteins have been proposed for such a role on the basis of genetic and biochemical evidence. In this work we show that the human Pbxl and HOXBl proteins can cooperatively activate transcription through a genetically charaterized Hox responsive element, the autoregulatory enhancer of the mouse Hoxb-T gene (b-lARE), which directs the spatially restricted expression of Hoxb-L in the fourth rhombomere during hindbrain development. On the b-lARE, only a restricted subset of HOX proteins is able to bind cooperatively and activate transcription. Selective recognition of the b-lARE is mediated by the N-terminal region of the HOX homeodomain. The DNA binding and protein-protein interaction functions of HOXBl and Pbxl are all necessary for the assembly of a transcriptionally active complex on the b-lARE. Functional dissection of the complex allowed the localization of the main activation domain in the HOXBl N-terminal region, and of an additional one in the C-terminal region of Pbxl, which is absent in one of its two alternative splicing isoforms. The transcriptional activity of HOX/Pbxl complexes on the b-lARE element is maximal and further restricted in embryonal carcinoma (EC) cells compared to other cell lines. Mutational analysis shows that an octamer-binding protein consensus site on the b-lARE contributes to the activity of the enhancer in transfected cells and it is bound in vitro by octamer-like proteins from both EC cells and embryonic extracts.