The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts, hoxb3a transcripts I-III and hoxb4a transcripts I-II, driven by four promoters. A "master" promoter, located about 2 kb downstream of hoxb5a, controls transcription of a pre-mRNA comprising exon sequences of both genes. This unique gene structure is proposed to provide a novel mechanism to ensure overlapping, tissue-specific expression of both genes in the posterior hindbrain and spinal cord. Transgenic approaches were used to analyze the functions of zebrafish hoxb3a/hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics. Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres (r) 5 to 8, suggesting a late role of the genes in neuronal cell lineage specification. Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6, whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5. We conclude that the similarity of hoxb3a/Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene.