ROMK (Kir1.1) low conductance, inwardly rectifying potassium channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. The regulation of ROMK channel function is complex and involves a multitude of factors that determine their open probability and their surface expression. It has long been known that pancreatic, cardiac and neuronal ATP-sensitive potassium channels couple energy metabolism with the electrical activity of the cells. Much less is known about the metabolic regulation of ROMK channels. We have studied the molecular nature of the ROMK channel protein complex in order to better understand its regulation. To identify novel regulatory subunits of the channel, we performed a membrane yeast two hybrid screen, using ROMK2 (Kir1.1b) as a bait. Screening a human kidney cDNA library, we identified two glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase-a as potential interacting partners. The interaction was verified by GST pulldown and co-localization assays. In addition, ROMK channels and the interacting enzymes were co-expressed in Xenopus oocytes and channel activity was studied by two-electrode voltage-clamp analysis. Surface expression of HA-tagged ROMK channels was determined with an antibody-based luminometric method. We found that GAPDH and enolase-a reduced the current flowing through ROMK channels but did not alter their surface expression. These results suggest that GAPDH and enolase-a form an integral part of the ROMK2 channel supramolecular complex and couple anaerobic glycolysis with channel activity.