The morphogenesis of the vertebrate hindbrain involves a transient segmentation process leading to the formation of reiterated organisation units called rhombomeres (r). A number of regulatory genes expressed with a rhombomere-specific pattern have been identified, including the gene encoding the transcription factor Krox-20, which is restricted to r3 and r5. We have previously demonstrated that in r3 and r5 Krox-20 directly controls the transcription of Hoxa-2 and Hoxb-2. In the present study, we provide evidence that Krox-20 is required for the expression of another Hox gene, Hoxb-3, in r5 specifically. Furthermore, the regulatory role of Krox-20 is not restricted to the control of Hox gene expression, since it is also involved in the activation of a receptor tyrosine kinase gene, Sek-1, in r3 and r5 and in the repression of the follistatin gene in r3 but not in r5. In conclusion, at least five regulatory genes belonging to different families are under the direct or indirect control of Krox-20 in r3 and/or r5 and this transcription factor therefore appears as a key regulator of gene expression in the developing hindbrain.