ATP-sensitive potassium (K_ATP) channels are known to be critical in the control of both insulin and glucagon secretion, the major hormones in the maintenance of glucose homeostasis. The involvement of K_ATPchannels in glucose uptake in the target tissues of insulin, however, is not known. We show here that Kir6.2(−/−) mice lacking Kir6.2, the pore-forming subunit of these channels, have no K_ATPchannel activity in their skeletal muscles. A 2-deoxy-[³H]glucose uptake experiment in vivo showed that the basal and insulin-stimulated glucose uptake in skeletal muscles and adipose tissues of Kir6.2(−/−) mice is enhanced compared with that in wild-type (WT) mice. In addition, in vitro measurement of glucose uptake indicates that disruption of the channel increases the basal glucose uptake in Kir6.2(−/−) extensor digitorum longus and the insulin-stimulated glucose uptake in Kir6.2(−/−) soleus muscle. In contrast, glucose uptake in adipose tissue, measured in vitro, was similar in Kir6.2(−/−) and WT mice, suggesting that the increase in glucose uptake in Kir6.2(−/−) adipocytes is mediated by altered extracellular hormonal or neuronal signals altered by disruption of the K_ATPchannels.