The spatially restricted expression of mammalian homeobox genes in teh embryonic central nervous system (CNS) provides an opportunity to study the basis of spatial gene regulation in mammalian development. Here, we define a regulatory region of the murine Hox 1.3 gene that mediates such a region-specific expression pattern. The Hox 1.3 gene contains two exons, encodes a putative protein of 270 amino acids, and is expressed preferentially in the spinal cord at midgestation. We have analyzed transgenic mice containing various Hox 1.3 DNA fragments fused to reporter sequences, such as a human growth hormone gene fragment or the E. coli lacZ structural gene. As shown by RNAase protection assays or by in situ analyses of beta-galactosidase activity, several hybrid genes are expressed in the embryonic central nervous system in a spatially restricted manner, along both the rostrocaudal and dorsoventral axes. A 912 nucleotide sequence located immediately upstream of the Hox 1.3 coding sequence is sufficient to direct expression to the dorsolateral cells of the brachial spinal cord.