The apical membrane Na⁺/H⁺ antiporter isoforms, NHE-2 and NHE-3, are involved in transepithelial Na⁺ absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate the NHE2 promoter expression in C2/bbe cells. A small DNA region (-85/+249) is involved in regulation of the basal transcriptional activity of the NHE-2 promoter as determined by transient transfection assays. RT-PCR analysis showed that the NHE2 mRNA was up-regulated in response to PMA. Results from actinomycin D treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in the promoter activity through elements located on -415/+249 DNA fragment. A PMA-induced nuclear factor that bound to NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA-response elements in the -415/+1 promoter region that bind to Sp1 and Sp3 in untreated and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.