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