Guanylyl cyclase C (GC-C), specifically expressed by intestinal epithelial cells, is the receptor for the Escherichia coli heat-stable enterotoxin that causes diarrhea. Tissue-specific expression of GC-C is mediated by the intestinal transcriptional regulator CDX2. This trans-activating protein regulates intestine-specific expression by binding to a critical sequence in the proximal promoter of GC-C. The precise nucleotide elements mediating CDX2 binding to promoter elements remain undefined. Several nuclear proteins form complexes with a DNA probe containing the promoter element of GC-C mediating CDX2 binding. The present study examined the nucleotide requirements in the consensus binding site and flanking regions in the cis element that mediates specific CDX2 binding to the promoter of GC-C. These studies identified seven core base pairs in the critical promoter element mediating tissue-specific expression of GC-C that are required for CDX2 binding. In addition, base pairs flanking this core sequence contribute to and are required for CDX2 recognition. These studies describe the precise nucleotide sequence within the GC-C promoter that comprises the CDX2 binding site required for intestine-specific expression.