A rapid filtration method was used to measure initial rates of 3-O-[3H]methylglucose uptake and thus estimate hexose transport system activity in isolated white fat cells. Insulin markedly stimulated the transport system activity and its effect was rapidly and completely reversible. In addition, such oxidants as vitamin K5 (50 muM), hydrogen peroxide (4mM), methylene blue (50 muM), and diamide (20 mM) also maximally activated 3-O-methylglucose transport and their effects were not additive to those of maximal concentrations of insulin. These oxidants had no effect on total cellular ATP levels under these conditions. Hexose transport system activity in either the presence or absence of these stimulatory agents was uniformly sensitive to inhibition by cytochalasin B. Treatment of fat cells with either 0.5 mM N-ethylmaleimide or 3 mM dithio(bis)nitrobenzoic acid abolished the ability of insulin or oxidants to activate hexose transport system activity. Control transport activity was not significantly influenced by these agents. Fat cells treated with dithio(bis)nitrobenzoic acid completely regained the ability to respond to insulin or vitamin K5 after removal of the agent by washing in low concentrations of reductant. Elevated rates of transport due to prior incubation of cells with insulin or vitamin K5 were completely resistant to inhibition by subsequent addition of N-ethylmaleimide or dithio(bis)nitrobenzoic acid. Deactivation of the hormone-stimulated transport system could be achieved by washing cells free of insulin or by destruction of insulin-receptor interaction by trypsin. N-Ethylmaleimide effectively blocked deactivation of insulin-stimulated transport system activity, while dithio(bis)nitrobenzoic acid was without effect. These results suggest that distinct cellular components mediate activation versus deactivation of the fat cell hexose transport system. N-Ethylmaleimide, which effectively penetrates fat cells, inhibits both processes while the layer, more polar dithio(bis)nitrobenzoic acid blocks activation but not deactivation of this transport system.