The regulation of ion channels by anionic phospholipids is currently very topical. An outstanding issue is whether phosphatidylinositol 4,5-diphosphate and related species act as true second messengers in signaling or behave in a manner analogous to an enzymatic cofactor. This question is especially pertinent regarding ATP-sensitive K+ channels in smooth muscle, for which there is substantial literature supporting inhibitory regulation by hormones. In this study, we have examined regulation of the potential cloned equivalents of the smooth muscle ATP-sensitive K+ channel (SUR2B/Kir6.1 and SUR2B/Kir6.2). We find that both can be inhibited via the Gq/11-coupled muscarinic M3 receptor but that the pathways by which this occurs are different. Our data show that SUR2B/Kir6.1 is inhibited by protein kinase C and binds anionic phospholipids with high affinity, such that potential physiological fluctuations in their levels do not influence channel activity. In contrast, Kir6.2 is not regulated by protein kinase C but binds anionic phospholipids with low affinity. In this case, phosphatidylinositol 4,5-diphosphate and related species have the potential to act as second messengers in signaling. Thus, Kir6.1 and Kir6.2 are regulated by distinct inhibitory mechanisms.