We sought to clone and characterize the murine Hox-b3 gene. In Xenopus embryos, the homologous gene has been shown to be responsive to retinoic acid, an agent which has profound effects on tissue growth and development. By plaque hybridization, using a partial, murine Hox-b3 cDNA as a probe, we screened a genomic library and isolated a series of overlapping clones. Restriction fragments from positive clones were sequenced by the dideoxy method on an automated DNA sequencer. We report the genomic sequence of the murine Hox-b3 gene. The sequence has been submitted to the GenBank database (accession number U02278). Our sequence extends from the P1 promoter through the coding sequence of the gene to the 3'-untranslated region. In common with other homeobox genes, there is an intron between the conserved hexapeptide and the homeobox. It is 866 bp long and has 3'- and 5'-splice sites very similar to the consensus, a long polypyrimidine tract and a potential branch point near the 3'-splice site. We have analyzed the sequence 5' to the initiation codon and the intron for putative control elements, and have identified a series of putative transcription factor binding sites in the P1 promoter and intron, including two for the retinoid X receptor-beta. Their possible significance is discussed. The sequences we have identified may be responsible for the observed pattern of expression of the gene. This sequence and the clones from which it is derived will enable a molecular dissection of the P1 promoter region.