The ROMK (Kir1.1) low conductance, inwardly rectifying, ATP-sensitive potassium channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. The regulation of ROMK channel activity is complex and involves a multitude of factors that determine its open probability. To identify novel regulatory subunits of the ROMK channel complex, 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 a GST pulldown assay. ROMK channels were expressed in Xenopus oocytes, and the effect of co-expressed GAPDH on channel activity and surface expression of the channel were determined by two-electrode voltage-clamp analysis and a quantitative surface expression assay. The functional relevance of our findings is demonstrated by the ability of GAPDH and enolase-a to partially block ROMK2 channel currents without influencing its surface trafficking. Co-expression of the glycolytically inactive GAPDH mutant, C149G did not have any effect on ROMK2 current amplitude or surface expression. Taken together, our results suggest that, GAPDH and enolase-a form an integral part of the ROMK2 channel macromolecular complex, coupling glycolysis with channel activity.