The Hox homeobox genes cooperate in providing positional information needed for spatial and temporal patterning of the vertebrate body axis. However, the biological mechanisms behind spatial Hox expression are largely unknown. In transgenic mice, gene fusions between Hoxa-5 (previously called Hox-1.3) 5' flanking regions and lacZ show tissue- and time-specific expression in the brachial spinal cord in day 11-13 embryos. This spatially-specific expression is directed by a 604 bp regulatory region with enhancer properties. Fine-detail mapping of this enhancer has identified several elements involved in region-specific expression. A brachial spinal cord (BSC) element, is required for expression in the brachial spinal cord and the upper cervical repressor (UCR) element is required to repress expression in the upper cervical region of the spinal cord. Factors in embryonic day 12.5 nuclear extracts bind these elements in electrophoretic mobility shift assays (EMSA). Factors binding the BSC element protect three regions from DNase digestion. All three sites contain an AAATAA sequence and mutations at these sites reduce or abolish binding. Furthermore, this element binds specific individual embryonic proteins on a protein blot. The binding appears as a gradient along the A-P axis with two- to three-fold higher levels observed in extracts from anterior regions than from posterior regions. In parallel with the EMSA, the proteins on the protein blot also show reduced binding to probes with mutations at the AAATAA sites. Binding activity to both of these elements is temporally-specific. Strong binding is present from early to midgestation but absent prior to birth. We also show binding activity to the BSC element in the same tissues in which Hoxa-5 is specifically expressed and that this binding activity is not inducible by nerve growth factor. These elements and the factors specifically-binding them are, therefore, likely to be involved in regulating the spatial-specific expression of Hoxa-5. This brachial spinal cord element and its binding proteins are likely to be involved in spatial expression of Hoxa-5 during development.