HOX genes encode highly conserved transcription factors responsible for developmental patterning and postnatal tissue homeostasis. Previous studies have shown that a 1.4-kb segment of the Hoxb-7 proximal promoter drives renal expression of reporter genes specifically in the ureteric bud and collecting ducts. In this study using stably transfected renal tubule cell lines, we have identified three short cis-acting sequences within this promoter segment that cooperate to induce high-level expression specifically in collecting duct cells. In addition to an inverted CCAAT box (-71/-67) that acts as an ubiquitous enhancer and binds the transcription factor CBF/NF-Y, two different cis-acting sequences, named CDSE-1 and CDSE-2 (for Collecting Duct Specific Element 1 and 2), allow collecting duct cell-specific promoter activation. CDSE-1 (-56/-34) is composed of two E-boxes separated by a 9-bp GC-rich sequence. Only the latter sequence enhances reporter gene expression specifically in collecting duct cells. CDSE-2 (-34/-13) contains sequence bears high homology with a segment of the Pax-2 promoter. CDSE-2 also conveys cell specificity but has no enhancer activity by itself.