Fat cells isolated from the epididymal (E) and dorsal subcutaneous (S) depots from 150g male Wistar rats were similar in size, but differed markedly in glucose metabolism. Retroperitoneal (RP) fat cells were slightly larger but were metabolically similar to epididymal fat cells. Basal incorporation of [1-14C] glucose into fatty acids was lower in S than E and RP, CO2 production and glyceride-glycerol synthesis were similar in all three; and lactate production was increased in RP and S compared to E. S adipocytes exhibited a blunted respond to both submaximally and maximally-stimulating concentrations of insulin compared to RP and E adipocytes in glucose incorporation into fatty acids, CO2 and lactate production, but not glyceride-glycerol. Maximally insulin-stimulated fatty acid synthesis by S fat cells was 19% and 29% of the values in E and RP fat cells respectively. Basal and maximally insulin-stimulated glucose transport (2-deoxy [14C] glucose uptake) was depressed by 30%-40% in S cells compared to E and RP. Thus, the decreased basal glucose utilization of S could be attributed primarily to a decreased glucose transport capacity. The markedly lower insulin-stimulated glucose metabolism in S fat cells, however, may be explained by alterations of the capacities for both transport and intracellular metabolism. Subcutaneous fat cells were also somewhat less sensitive to submaximal doses of insulin and this was reflected in rightward shift in the dose-response curves for 2-deoxyglucose uptake and fatty acid synthesis. The decreases in insulin stimulated glucose oxidation and fatty acid synthesis were paralleled by decreases in the major lipogenic enzymes. Although the reason for these variations in the capacity for glucose metabolism among depots is unknown, they are important in assessing the metabolic function of the whole adipose organ.