Palmitate has been shown to stimulate glucose transport, translocation of GLUT4 and insulin receptor autophosphorylation in isolated rat adipocytes (Biochem Biophys Res Commun 177:343-49, 1991). Here we further characterize the ability of short-term treatment with free fatty acids to stimulate glucose transport in isolated rat adipocytes and demonstrate that prolonged treatment induces insulin resistance. Treatment of adipocytes for 15 min with 1 mM myristate (14:0), palmitate (16:0), or stearate (18:0) stimulates glucose transport by 119 +/- 33, 89 +/- 29, and 114 +/- 30%, respectively. In contrast, oleate (cis 18:1), 1), elaidate (trans 18:1), and linoleate (cis 18:2) do not stimulate glucose transport. Palmitate stimulates glucose transport in a concentration-dependent manner, demonstrating saturation at 1 mM and half-maximal stimulation at 0.25-0.5 mM. Prolonged treatment (4 h) of rat adipocytes with 1 mM palmitate induces insulin resistance. After a 4-h preincubation with palmitate (1 mM), insulin stimulates glucose transport in rat adipocytes by 4.4-fold +/- 0.8, vs. 8.8-fold +/- 0.8 in controls (n = 3). Palmitate-induced resistant cells demonstrated a 40% inhibition in maximal insulin responsiveness with little change in insulin sensitivity. Insulin binding is only slightly decreased (8%) in palmitate-pretreated cells. These studies indicate that saturated fatty acids stimulate glucose transport acutely and on prolonged exposure induce insulin resistance via a post-insulin binding defect. The underlying molecular mechanisms of insulin resistance induced by prolonged treatment with saturated fatty acids may now be investigated using this unique cellular model.