Because little is known about the molecular basis of transcriptional regulation of the murine H+-K+-ATPase α2(HKα2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HKα2promoter-reporter gene construct. In these mice, the region −7,264/+253 of the HKα25′-flanking region controls expression of the reporter gene enhanced green fluorescent protein (EGFP). Patterns of HKα2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of 10 major organs examined, EGFP immunoreactivity was detected abundantly in the kidney, and to a far lesser extent, in the brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of transgenic mice and colocalized with the cellular distribution of both endogenous HKα2and aquaporin-2, consistent with the known expression pattern of endogenous HKα2in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of the highest endogenous HKα2expression. Although previous studies of steady-state mRNA levels suggested differences in HKα2gene regulation in the kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HKα2gene in these organs and suggest that regions outside the 5′-flanking region or other regulatory factors play a role in HKα2expression in the distal colon.