Our earlier studies have shown that Kir2.x channels are suppressed by an increase in the level of cellular cholesterol whereas cholesterol depletion enhances the activity of the channels. In this study, we show that Kir2.1 and Kir2.3 channels have double peak distributions between cholesterol-rich (raft) and cholesterol-poor (non-raft) membrane fractions indicating that the channels exist in two different types of lipid environment. We also show that while MCD-induced cholesterol depletion removes cholesterol from both raft and non-raft membrane fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions. Kinetics of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlates with the changes in the level of raft cholesterol. Furthermore, we show not only that cholesterol depletion shifts the distribution of the channels from cholesterol-rich to cholesterol-poor membrane fractions but also that cholesterol enrichment has the opposite effect. These observations suggest that change in the level of raft cholesterol alone is sufficient to suppress Kir2 activity and to facilitate partitioning of the channels to cholesterol-rich domains. Therefore, we suggest that partitioning to membrane rafts plays an important role in the sensitivity of Kir2 channels to cholesterol.