We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37°C) and cell surface insulin binding (16°C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (K(i) = 2.2 ± 0.04 mM) compared with maximal transport rates (K(i) = 4.1 ± 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect. In studies examining the mechanism of decreased glucose transport in maximally-stimulated cells, the number of D-glucose-inhibitable cytochalasin B binding sites was reduced in the plasma membrane by D-glucose plus insulin pretreatment (9 ± 2 pmol/mg protein compared with 18 ± 1 in controls or 16 ± 2 with insulin alone) and increased in low density microsomes (36 ± 4 compared with 26 ± 7 in controls or 23 ± 5 insulin alone); however, the number of cellular binding sites in a total membrane fraction was similar in these binding groups. In conclusion, 1) intracellular metabolism of glucose modulates insulin's long-term ability to regulate glucose transport system activity, and 2) D-glucose plus insulin decrease insulin responsiveness by impairing translocation of glucose transporters to the cell surface and not by reducing the total number of cellular glucose transporters. This mechanism of insulin resistance may be relevant to the formation of cellular defects in non-insulin-dependent diabetes mellitus.