Early brain regionalisation involves the activation of genes coding for transcription factors in distinct domains of the neural plate. The limits of these domains often prefigure morphological boundaries. In the hindbrain, anteroposterior patterning depends on a segmentation process that leads to the formation of seven bulges called rhombomeres (r). The molecular cues involved in the early subdivision of the hindbrain and in rhombomere formation are not well understood. We show that iro7, a zebrafish gene coding for a transcription factor of the Iroquois family, is expressed at the end of gastrulation in the future midbrain and hindbrain territories up to the prospective r4/r5 boundary. This territory is strictly complementary to the expression domain of another homeobox gene, vhnf1, in the caudal neural plate. We demonstrate that Iro7 represses vhnf1 expression anterior to their common border and that, conversely, vHnf1 represses iro7 expression caudal to it. This suggests that the r4/r5 boundary is positioned by mutual repression between these two transcription factors. In addition, iro7 is involved in the specification of primary neurons in the rostral hindbrain. In particular, it is essential for the formation of the Mauthner neurons in r4. We propose that iro7 has a dual function in the hindbrain of the zebrafish embryo: it is required for the proper positioning of the prospective r4/r5 boundary and it promotes neurogenesis in the anterior hindbrain.