To examine the possible role of homeo box genes in murine development we have studied the structure and expression pattern of Hox-2.5, a newly isolated homeo box gene that maps to the left end of the mouse Hox-2 locus on chromosome 11. The sequence of the Hox-2.5 homeo box has been determined. It is highly homologous to Hox-1.7 and Hox-3.2, demonstrating extended conservation among three homeo box complexes in the mouse. Northern and in situ hybridization analyses of Hox-2.5 demonstrate a novel, regionally restricted pattern of expression in developing mesoderm and neurectoderm. We detect localized Hox-2.5 transcripts as early as 8.5 days postcoitum. The expression pattern of Hox-2.5 was analyzed over the next 3 days of ontogeny, as well as in later embryonic, newborn, and adult stages. Three-dimensional reconstruction of Hox-2.5 transcript localization within the central nervous system of early embryos clearly illustrates the neural expression domain. Although the Hox-2.5 expression pattern is regionally restricted during all of these stages of development, the pattern changes along the anteroposterior and dorsoventral axes of the CNS as the embryo undergoes complex morphogenetic movements and cytodifferentiation.