The AMP-activated serine/threonine protein kinase (AMPK) is activated by energy depletion, increase in cytosolic Ca²⁺ activity, oxidative stress and nitric oxide. AMPK participates in the regulation of the epithelial Na⁺ channel ENaC and the voltage-gated K⁺ channel KCNE1/KCNQ1. The present study explored whether AMPK regulates the renal outer medullary K⁺ channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK^gR70Q (AMPKa1+AMPKb1+AMPKg1^R70Q), or of inactive AMPK^aK45R ((AMPKa1^K45R+AMPKb1+AMPKg1)), and the current determined utilizing two-electrode voltage-clamp. Moreover, renal Na⁺ and K⁺ excretion were determined in AMPKa1-deficient mice (ampk^-/-) and wild type mice (ampk^+/+) prior to and following an acute K⁺ load (in mM: 111 KCl, 30 NaHCO₃, 4.7 NaCl, 2.25 g/dl BSA) at a rate of 250 ml/h. As a result, coexpression of AMPK^gR70Q but not of AMPK^aK45R significantly decreased the current in ROMK-expressing Xenopus oocytes. Under control conditions, no significant differences were observed in plasma Na⁺ and K⁺ concentration as well as in absolute and fractional Na⁺ and K⁺ excretions between ampk^-/- and ampk^+/+ mice. Following an acute K⁺ load, plasma Na⁺ and K⁺ concentration were again similar in both genotypes, but absolute and fractional Na⁺ and K⁺ excretion were higher in ampk^-/- than in ampk^+/+ mice. In conclusion, AMPK down-regulates ROMK, an effect compromising the ability of the kidney to excrete K⁺ following an acute K⁺ load.