Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and gamma-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.