The Hox genes have been proved to be instrumental in establishing the positional identity of cells along the embryonic anteroposterior (A-P) axis. Studying the regulation of these genes is a first step toward elucidating the molecular basis of regionalization during embryogenesis. We report here on the identification of cis-acting elements controlling the expression of Hoxb-7 (Hox-2.3). We show that elements driving A-P restricted gene expression are located within the 3.5 kb proximal upstream sequences of the Hoxb-7 gene. A deletion analysis provides evidence for at least three cis-acting control elements upstream from Hoxb-7, and for cooperative interactions between some of these elements in generating the A-P restricted transgenic pattern. One element, conferring by itself Hox-like expression boundaries to the transgene, has been studied in more detail and found to act in an orientation-and promoter-dependent manner. Together the 3.5 kb sequences proximal to Hoxb-7 mediate A-P restricted Hoxb-7/lacZ gene expression in a domain showing rostral boundaries more posterior than those of Hoxb-7. The evolution throughout embryogenesis of the expression pattern of a transgene carrying these sequences has been analysed and shown to mimick that of the endogenous gene, except for a slight delay in the initial expression. We conclude that the transgenes that we tested, spanning a total of 27 kb genomic sequences, do not reproduce all the features of the Hoxb-7 expression pattern. The differences in expression between Hoxb-7 and the transgenes may reveal an aspect of the Hox regulation for which either remote cis-acting control elements and/or gene clustering is required. Additional features that may have favoured maintenance of clustered organisation during evolution are partial overlap of transcription units with the regulatory regions of the neighbouring genes, and cis-regulatory interactions between multiple Hox genes: not only do cis-acting control elements of the Hoxb-7 gene map in the 3' untranslated sequences of the Hoxb-8 (Hox-2.4) gene, but our experiments suggest that Hoxb-7 control sequences modulate expression of the Hoxb-8 gene as well.