The epithelial Na+ channel (ENaC) was previously shown to be expressed in several Na(+)- and fluid-absorbing epithelia, particularly those of the kidney, colon, and lung. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein with ENaC and demonstrated that Nedd4 binds by its WW domains to the proline-rich PY motifs of ENaC. These PY motifs were recently shown to be deleted/mutated in patients afflicted with Liddle's syndrome, a hereditary form of systemic renal hypertension. Such mutations cause elevated channel activity by an increase in channel number/stability at the plasma membrane and by increased channel opening. We then proposed that Nedd4, by regulating channel stability/ degradation, may be a suppressor of ENaC. To test whether Nedd4 is localized to those tissues/regions that express ENaC, we performed immunocytochemical analysis of rat Nedd4 (rNedd4) distribution in rat kidney, colon, and lung tissues. Our results show that, in the kidney, rNedd4 is primarily localized to the cortical collecting tubules and outer and inner medullary collecting ducts. These tubular segments were previously shown to express ENaC. The epithelium lining medullary calyxes was also intensely stained, and microvillar borders of proximal convoluted tubules expressed variable amounts of rNedd4. In the lung, rNedd4 was mainly expressed in the epithelia lining the airways, in the submucosal glands and ducts, and in the distal respiratory epithelium. These sites resemble the pattern of ENaC expression. In contrast, in the distal colon, rNedd4 was strongly expressed in the epithelia lining the crypts but not in the ENaC-expressing surface epithelium. Low-salt diet (to elevate serum aldosterone levels) had no effect on rNedd4 distribution in the kidney or colon. Thus Nedd4 is coexpressed and likely colocalizes with ENaC in specific regions within the kidney and lung but not in the colon. We speculate this difference in colocalization may reflect differences in the regulation of channel stability in those tissues.