Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VAMP 2 and syntaxin 4. Microinjection of tetanus toxin light chain or botulinum D toxin light chain resulted in an 80 and 61% inhibition respectively of insulin stimulation of GLUT4 translocation in 3T3L1 cells assessed using the plasma-membrane lawn assay. Botulinum A toxin light chain, which cleaves SNAP-25, was without effect. Microinjection of an N-terminal VAMP 2 peptide (residues 1–26) inhibited insulin stimulation of GLUT4 translocation by 54%. A syntaxin 4 peptide (residues 106–122) inhibited insulin stimulation of GLUT4 translocation by 40% whereas a syntaxin 1c peptide (residues 226–260) was without effect. These data taken together strongly suggest a role for VAMP 2 in GLUT4 trafficking and also for syntaxin 4. They further indicate that the isoforms of SNAP-25 isolated to date that are sensitive to cleavage by botulinum A toxin light chain do not appear to be involved in GLUT4 translocation.