Claudins, and particularly claudin‐2, are important regulatory components of tight junction permeability. A better understanding of the involvement of claudin‐2 in intestinal barrier functions requires the characterization of its distribution and regulation in the intestine. Interestingly, the claudin‐2 gene promoter harbors a number of similarities to that of sucrase‐isomaltase, a marker of enterocyte differentiation. We thus investigated the expression of claudin‐2 in relation to the transcription factors CDX2, HNF‐1α, and GATA‐4 in the human intestine. The characterization of claudin‐2 and the expression of the above transcription factors were performed by immunofluorescence, Western blot, and RT‐PCR in the developing human intestinal epithelium. The functional role of CDX2, HNF‐1α, and GATA‐4 on claudin‐2 regulation was also examined by ectopic expression studies in intestinal cell models. Claudin‐2 was detected in both crypt and villus cells of the small intestine but restricted to undifferentiated crypt cells in the colon. CDX2 and HNF‐1α were expressed along the entire intestine whereas GATA‐4 was undetectable in the colon. Accordingly, in the colonic Caco‐2 cell model, claudin‐2 was found to be present only in undifferentiated cells. Like in the colonic epithelium, GATA‐4 was found to be also lacking in Caco‐2 cells while CDX2 and HNF‐1α were present at significant levels. Cotransfection experiments showed that the claudin‐2 promoter was activated by CDX2, HNF‐1α, and GATA‐4 in a cooperative manner. Furthermore, forced GATA‐4 expression in Caco‐2 cells enhances maintenance of claudin‐2 expression during differentiation. These observations suggest that optimal claudin‐2 expression in the gut relies on the presence of GATA‐4, suggesting a role for this factor in intestinal regionalization. © 2004 Wiley‐Liss, Inc.