Glucose uptake and free fatty acid release by adipocytes are increased by catecholamines. The mechanism of the stimulatory action of catecholamines on glucose uptake may be via eicosanoid production from release fatty acids. Rats were fed iso-nutrient diets with high (HSO, 20% Kcal of the total Kcal) or low (LSO, 2% Kcal of the total Kcal) safflower oil. After one month, 5 rats per diet group were fed diets with aspirin (ASP group, 50 mg/Kg bw/day) or without aspirin (CON group) for 2 days. Isolated adipocytes from epididymal fat pads were incubated at 37°C, gassed with 95% O[subscript]2-5% CO[subscript]2 in KRB buffer with 3% bovine serum albumin and with or without eicosanoid modifiers; a stimulator (10[superscript]-5 M norepinephrine, N), or inhibitors (167 [mu]l of antiserum to prostaglandin E (AntiE) per 1,600 [mu]l or 23mM Asp), or combinations of these. At 2-, 5-, and 10-min of incubation, samples of incubation mixtures were taken to measure 2-deoxy glucose (2-DG) (a non-metabolizable D-glucose analog which is transported into adipocytes by the same process as D-glucose is) transport using [superscript]3H-2-deoxy glucose, [superscript]14C-inulin, and liquid scintillation counter. At 0- and 15-min incubation time, free fatty acids (FFA) were analyzed by gas chromatography after extraction with Dole reagent. Fatty acid composition of the fat pad was also measured by gas chromatography. Experiments were repeated four times. Amounts of dietary linoleic acid were directly reflected in the amounts of linoleic acid released into adipocyte incubation mixtures which were correlated with 2-DG transport by adipocytes (r = +0.60 in HSO-CON, +0.61 in LSO-CON; p < 0.009). HSO significantly (P ≤ 0.05) increased 2-DG transport by adipocytes compared with the LSO (8.66 vs. 6.31 nanomoles 2-DG transport per 10[superscript]6 cells at the 5 min incubation time). AntiE or Asp treatment in vitro significantly decreased 2-DG transport compared to controls in vitro. These results suggest that dietary linoleic acid has a regulatory effect on adipocyte glucose transport which is mediated by eicosanoids. Prostaglandin E seems to be particularly involved.