Long chain saturated fatty acids (LCSFA) cause insulin resistance in animal models. Although this resistance is ultimately characterized by decreased GLUT4 expression in adipocytes, the events leading to decreased GLUT4 remain unknown. Using an in vitro model of fatty acid-induced insulin resistance in rat adipocytes we have investigated early events in LCSFA-induced insulin resistance. In adipocytes made insulin resistant with 1 mM palmitate, insulin-stimulated GLUT4 translocation to the plasma membrane was unaffected (control 9.0+/-1.8 fold, palmitate 8.2+/-1,5) as was tyrosine phosphorylation of the insulin receptor (control 2.7+/-1.3 fold, palmitate 4.6+/-1.8 fold). Despite this, insulin-stimulated glucose transport was decreased in palmitate treated cells (37+/-6%). Insulin stimulated glucose transport measurements in isolated plasma membranes obtained from palmitate treated adipocytes also demonstrated insulin resistance (50-100% decrease). This decrease in plasma membrane glucose transport could be due to inhibition of the exofacial presentation or activation stages of insulin stimulated GLUT4 mediated glucose transport. Trypsinization of intact adipocytes and analysis of the GLUT4 cleavage products by immunoblot indicate that there was no difference in the exofacial expression of GLUT4 between fatty acid-treated and control adipocytes (p>0.05, n=3 separate experiments). These experiments indicate that the initial defect in saturated fatty acid induced insulin resistance in rat adipocytes is most likely due to an inhibition of GLUT4 activation.