Hox genes play a key role in the specification of regional development in the vertebrate embryo. They are expressed in regionally restricted domains along the anterior-posterior axis, generally extending from a sharp rostral boundary toward the posterior end. We have studied the regulation of the murine Hoxb-8 gene in vivo using reporter constructs and found that 11 kb of genomic sequences upstream of Hoxb-8 confer a Hox-like pattern of expression on a lacZ reporter gene fused in-frame to the first exon of Hoxb-8. Reporter gene expression was detectable from early stages onwards, but reached rostral expression boundaries in mesoderm and neurectoderm that were more posterior than those of the endogenous gene. Within the upstream region, we have identified several cis-acting elements which are individually capable of driving regionally restricted expression in combination with the Hoxb-8 promoter, and we have investigated their relative contributions to the expression pattern. The results suggest that, in this experimental context, these upstream elements, as well as previously identified elements located within the Hoxb-8 gene, cooperate in setting the rostral expression boundaries. Furthermore, we show that multiple identical copies of cis-acting elements can cooperate, causing a pronounced anterior shift in the expression boundaries. This indicates that the rostral extent of expression is limited by the activity of a transcription factor binding to these elements and that this activity was, at some point in development, higher in precursors of more posterior structures than in precursors of anterior structures. This factor is thus very likely to be involved in the transduction of positional signals.