The AMP-activated protein kinase (AMPK) is a key controller of cellular energy metabolism. We studied its expression and regulation by salt handling in the kidney. Immunoprecipitation and Western blots of protein lysates from whole rat kidney using subunit-specific antibodies showed that the alpha1-catalytic subunit is expressed in the kidney, associated with the beta2- and either gamma1- or gamma2-subunits. Activated AMPK, detected by immunohistochemical staining for phospho-Thr172 AMPK (pThr172), was expressed on the apical surface of the cortical thick ascending limb of the loop of Henle, including the macula densa, and some parts of the distal convoluted tubule. Activated AMPK was also expressed on the basolateral surface of the cortical and medullary collecting ducts as well as some portions of the distal convoluted tubules. AMPK activity was increased by 25% in animals receiving a high-salt diet, and this was confirmed by Western blotting for pThr172. Low-salt diets were associated with reduced levels of the alpha-subunit of AMPK, which was highly phosphorylated on Thr172. Surprisingly, both low- and high-salt media transiently activated AMPK in the macula densa cell line MMDD1, an effect due to changes in osmolality, rather than Na+ or Cl- concentration. This study, therefore, demonstrates regulation of AMPK by both a high- and a low-salt intake in vivo and suggests a role for the kinase in the response to changes in osmolality within the kidney.