Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na(+) transport, effects at least partially involving regulation of the epithelial Na(+) channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na(+) channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function (V617F)JAK2 or inactive (K882E)JAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, (V617F)JAK2 or (K882E)JAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I(amil)) in dual electrode voltage clamp. Moreover, I(amil) was determined in colonic tissue utilizing Ussing chambers. As a result, the I(amil) in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 and Nedd4-2 decreased I(amil) in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I(amil). In colonic epithelium, I(amil) was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.