Homeobox genes are expressed in stage-, region-, and tissue-specific patterns during embryonic development of the mouse. In order to understand the underlying regulatory mechanisms the murine homeobox gene Hoxb-6 was analyzed for the presence of cis-acting regulatory elements. Transgenic mouse embryos and lines were generated which contained the LacZ reporter gene under the control of different fragments from the Hoxb-6 gene. In total, 13.2 kb of genomic DNA covering the entire Hoxb-6 region were analyzed. Our results suggested the presence of three regulatory regions in the Hoxb-6 gene: a limb/LPM element which directed gene expression into restricted regions of the developing limb buds and the ventro-lateral and visceral mesenchyme, a regulatory element required for gene expression into ventral regions of the developing spinal cord, and a third element necessary for directing gene expression into developing mesonephric tubules and mesonephric ducts. We demonstrated that the limb/LPM element functions as an enhancer in a promoter- and orientation-independent manner. The comparison of the expression patterns of the reporter gene constructs and the endogenous Hoxb-6 gene revealed that the regulatory regions were able to reproduce part of the Hoxb-6 pattern. However, not all control elements necessary to completely mimic the endogenous Hoxb-6 expression pattern could be detected, although the entire Hoxb-6 genomic region has been analyzed. These observations suggest that for some aspects of Hoxb-6 gene regulation the integrity of large genomic regions of the Hoxb cluster is required.