Glucose utilization was studied in isolated adipocytes from rats fed a mixed (51% carbohydrate, 30% fat, 19% protein), high fat (81% fat, 19% protein) or high protein diet (30% fat, 70% protein). Despite similar food intake, rats on the high protein (HP) diet had smaller epididymal fat pads than the other two groups. The reduction in fat pad size was caused by small and variable reductions in both cell size and cell number. Fat cells from rats on the high fat (HF) diet had the previously reported reduction in pentose phosphate shunt activity in the absence and presence of insulin, and marked diminution of de novo fatty acid synthesis. Lactate release was elevated in the absence of insulin. There was no insulin stimulation of glucose uptake, CO2 production, glyceride-glycerol production or lactate release in these adipocytes. However, significant stimulation of fatty acid synthesis was seen. There was no impairment of glucose uptake or utilization in cells from rats on the HP diet despite the absence of dietary carbohydrate. Indeed, 14CO2 produced from glucose-l-14CO2 produced from glucose-1-14C was increased in these adipocytes. Thus the impaired glucose utilization in rats on the high fat, carbohydrate-free diet is due solely to the fat content of the diet.