Because little is known about the molecular basis of transcriptional regulation of the murine H⁺-K⁺- ATPase 2 (HK2) gene or other genes whose expression is restricted in part to the collecting duct, especially in vivo, we developed transgenic mice carrying an insertional HK2 promoter-reporter gene construct. In these mice, the region -7264/+253 of the HK2 5'-flanking region controls expression of the reporter gene EGFP. Patterns of HK2/EGFP transgene expression were examined by fluorescence microscopy and immunoblotting. Of ten major organs examined, EGFP immunoreactivity was detected abundantly in kidney, and to a far lesser extent in brain and lung. Within the kidney, EGFP fluorescence was detected exclusively in the collecting ducts of the transgenic mice, and colocalized with the cellular distribution of both endogenous HK2 and aquaporin-2, consistent with the known expression pattern of endogenous HK2 in principal cells. Surprisingly, no transgene expression was evident by immunoblotting or fluorescence microscopy in the distal colon, the site of highest endogenous HK2 expression. Though previous studies of steady-state mRNA levels suggested differences in HK2 gene regulation in kidney and colon, our results provide the first direct evidence of differential transcriptional control of the HK2 gene in these organs and suggest that regions outside the 5'-flanking region or other regulatory factors play a role in HK2 expression in the distal colon.