The mode of action of Hoxa1, like that of most Hox proteins, remains poorly characterized. In an effort to identify functional determinants contributing to the activity of Hoxa1 as a transcription factor, we generated 18 pentapeptide insertion mutants of the Hoxa1 protein and we assayed them in transfected cells for their activity on target enhancers from the EphA2 and Hoxb1 genes known to respond to Hoxa1 in the developing hindbrain. Only four mutants displayed a complete loss-of-function. Three of them contained an insertion in the homeodomain of Hoxa1, whereas the fourth loss-of-function mutant harbored an insertion in the very N-terminal end of the protein. Transcription activation assays in yeast further revealed that the integrity of both the N-terminal end and homeodomain is required for Hoxa1-mediated transcriptional activation. Furthermore, an insertion in the serine–threonine–proline rich C-terminal extremity of Hoxa1 induced an increase in activity in mammalian cells as well as in the yeast assay. The C-terminal extremity thus modulates the transcriptional activation capacity of the protein. Finally, electrophoretic mobility shift assays revealed that the N-terminal extremity of the protein also exerts a modulatory influence on DNA binding by Hoxa1–Pbx1a heterodimers. J. Cell. Biochem. 110: 484–496, 2010. © 2010 Wiley-Liss, Inc.