Elevated serum-free fatty acid (FFA) levels induce insulin resistance in whole animals and humans. To understand the direct mechanism by which FFAs impact insulin-responsive tissue, we have used our previously developed in vitro model of long-chain saturated fatty acids (LCSFA)-induced insulin resistance in adipocytes. In addition to explanted rat adipocytes, we now demonstrate that overnight exposure of 3T3-L1 adipocytes to 1 mm individually of the LCSFA palmitate, myristate, and stearate, leads to an approximately 50% inhibition of insulin-induced glucose transport. Insulin resistance can be accomplished at 0.3 mm palmitate, which is within the range of palmitate found in diabetic and obese individuals. This inhibition was noted within 4 h of exposure to FFA, which is comparable to in vivo lipid infusion studies. Initial LCSFA-induced resistance is specific to glucose transport and does not affect insulin stimulation of glucose incorporation into glycogen. In 3T3-L1 adipocytes overexpressing the EGF receptor, LCSFA exposure also specifically inhibited EGF-induced GLUT4-mediated glucose transport, but not EGF-induced glycogen synthesis. We find that LCSFA treatment did not impair insulin stimulation of GLUT4 translocation or exofacial presentation on the cell surface as determined by trypsin accessibility. Our results suggest that the initial direct effect of elevated LCSFA is to impair activation of GLUT4 transporter activity and that this effect is specific for glucose transport.