The mouse Hox-2.3 gene contains an Antp-like homeobox sequence and is expressed in a spatially restricted anteroposterior domain during development. To study the molecular basis of this differential gene regulation, we set out to characterize the cis-regulatory elements mediating Hox-2.3 expression during embryogenesis. We show that a fragment extending 1316 base pairs (bp) upstream of the transcription start site, thus corresponding to the Hox-2.4/Hox-2.3 intergenic sequences is capable of mediating luciferase gene transcription in transfected cells in vitro and lacZ expression in transgenic mice. The β-galactosidase-staining pattern in embryos was found to be strikingly similar to the Hox-2.3 in situ hybridization pattern in intermediate mesoderm derivatives: high levels of both Hox-2.3 transcripts and β-galactosidase activity were found in the mesonephric duct-derived epithelium of the meso- and metanephric kidney and associated ducts, from the time these structures first appeared on throughout development. The transgene apparently lacks sequences needed for correct Hox-2.3 expression in somitic and lateral plate mesoderm and in neurectoderm. These results document the involvement of distinct regulatory elements in Hox gene expression in subsets of cells with distinct developmental fate, situated at similar positions along the anteroposterior axis of the embryo.