Our aim was to determine the subcellular localization and functional roles of the K(ATP) channel subunit Kir6.1 in intracellular membranes. Specifically, we focused on the potential role of Kir6.1 as a subunit of the mitochondrial ATP-sensitive K+ channel. Cell imaging showed that a major proportion of heterologously expressed Kir6.1-GFP and endogenously expressed Kir6.1 was distributed in the endoplasmic reticulum with little in the mitochondria or plasma membrane. We used pharmacological and molecular tools to investigate the functional significance of this distribution. The K(ATP) channel opener diazoxide increased reactive oxygen species production, and glibenclamide abolished this effect. However, in cells lacking Kir6.1 or expressing siRNA or dominant negative constructs of Kir6.1, the same effect was seen. Ca2+ handling was examined in the muscle cell line C2C12. Transfection of the dominant negative constructs of Kir6.1 significantly reduced the amplitude and rate of rise of [Ca2+]( c ) transients elicited by ATP. This study suggests that Kir6.1 is located in the endoplasmic reticulum and plays a role in modifying Ca2+ release from intracellular stores.