Carbonic anhydrase 1 (CA1) is an abundant enzyme in colon epithelia. In the gastrointestinal tract, carbonic anhydrase is vital for NaCl resorption, alkalinization of gut contents, and absorption of short-chain fatty acids. The CA1 gene has two promoters, one of which is specifically active in colon epithelia and the other in erythroid cells. We are investigating the factors that regulate CA1 expression from the colon-specific promoter. Colon-specific deoxyribonuclease I hypersensitive sites (DHS) have been mapped close to the colon transcription initiation site (DHS6c) and in the upstream intron (DHS5c). Using electrophoretic mobility-shift assays to search the 650-bp region which contains DHS6c, we have identified sequences that bind a colon-specific factor (COF1) and by deletion analysis we have narrowed down the COF1-binding motif to a 17-bp sequence. A comparison of this motif with a protein-binding motif in the sucrase-isomaltase gene promoter, competition assays, and antibody studies indicate that COF1 is identical to the homeodomain protein Cdx-2. We propose that Cdx-2 plays an important role in the intestine-specific expression of CA1.