We examined, in soleus muscle, the effects of prolonged palmitate exposure (0, 6, 12, 18 h) on insulin-stimulated glucose transport, intramuscular lipid accumulation and oxidation, activation of selected insulin-signaling proteins, and the insulin-stimulated translocation of GLUT4. Insulin-stimulated glucose transport was progressively reduced after 6 h (-33%), 12 h (-66%), and 18 h (-89%) of palmitate exposure. These decrements were closely associated with concurrent reductions in palmitate oxidation at 6 h (-40%), 12 h (-60%), and 18 h (-67%). In contrast, intramuscular ceramide (+24%) and diacylglycerol (+32%) concentrations, insulin-stimulated AS160 (-36%) and PRAS40 (-33%) phosphorylations, and Akt (-40%), PKCtheta (-50%), and GLUT4 translocation (-40%) to the plasma membrane were all maximally altered within the first 6 h of palmitate treatment. No further changes were observed in any of these parameters after 12 and 18 h of palmitate exposure. Thus, the intrinsic activity of GLUT4 was markedly reduced after 12 and 18 h of palmitate treatment. During this reduced GLUT4 intrinsic activity phase at 12 and 18 h, the reduction in glucose transport was twofold greater compared with the early phase (< or =6 h), when only GLUT4 translocation was impaired. Our study indicates that palmitate-induced insulin resistance is provoked by two distinct mechanisms: 1) an early phase (< or =6 h), during which lipid-mediated impairments in insulin signaling and GLUT4 translocation reduce insulin-stimulated glucose transport, followed by 2) a later phase (12 and 18 h), during which the intrinsic activity of GLUT4 is markedly reduced independently of any further alterations in intramuscular lipid accumulation, insulin signaling and GLUT4 translocation.