High-fat diet (HFD) induces skeletal muscle insulin resistance. To investigate associated changes in the plasma membrane glucose transporter, male Sprague-Dawley rats were fed either chow [high-carbohydrate diet (HCD)] or HFD for 3 wk. Plasma membrane vesicles were prepared from hindlimb muscle of control, insulin-stimulated (Ins), and acutely exercised (Ex) rats. Maximal vesicle glucose transport activity (Vmax) increased threefold with Ins and Ex treatment compared with controls in HCD rats; in HFD rats, increases were less than twofold. Transporter numbers (measured by cytochalasin B binding, CB) approximately doubled with Ins and Ex in both diet groups. Intrinsic activity (carrier turnover, Vmax/CB) increased significantly with stimulation in HCD but not HFD rats. Therefore, vesicles from HFD rats showed resistance to both exercise and insulin stimulation of muscle glucose transport. Transporter number increased normally, but intrinsic activity in HFD rats did not respond. Two conclusions are discussed: 1) translocation and activation are distinct, separable steps in transporter stimulation and 2) HFD produces effects that resemble the insulin resistance of starvation.