In skeletal muscle, contractions increase glucose uptake due to a translocation of GLUT4 glucose transporters from intracellular storage sites to the surface membrane. Vesicle associated membrane proteins (VAMPs) are believed to play an important role in docking and fusion of the GLUT4 transporters at the surface membrane. However, knowledge about which VAMP isoforms in fact co-localize with GLUT4 vesicles in mature skeletal muscle and whether they translocate during muscle contractions is incomplete. The aim of the present study was to further identify VAMP isoforms which are associated with GLUT4 vesicles and examine which VAMP isoforms translocate to surface membranes in skeletal muscles undergoing contractions. VAMP2, VAMP3, VAMP5 and VAMP7 were enriched in immuno-precipitated GLUT4 vesicles. In response to 20 min of in situ contractions, there was a redistribution of GLUT4 (+64 +/- 13%), transferrin receptor (TfR; +75 +/- 22%) and insulin-regulated aminopeptidase (IRAP; +70 +/- 13%) to fractions enriched in heavy membranes away from low density membranes (-32 +/- 7%; -18 +/- 12%; -33 +/- 9%; respectively), when compared with the resting contralateral muscle. Similarly, there was a redistribution of VAMP2 (+240 +/- 40%), VAMP5 (+79 +/- 9%) and VAMP7 (+79 +/- 29%), but not VAMP3, to fractions enriched in heavy membranes away from low density membranes (-49 +/- 10%, -54 +/- 9%, -14 +/- 11%, respectively) in contracted versus resting muscle. In summary, VAMP2, VAMP3, VAMP5 and VAMP7 co-immuno-precipitate with intracellular GLUT4 vesicles in muscle and VAMP2, VAMP5, VAMP7, but not VAMP3, translocate to the cell surface membranes similar to GLUT4, TfR and IRAP in response to muscle contractions. These findings suggest that VAMP2, VAMP5 and VAMP7 may be involved in translocation of GLUT4 during muscle contractions. Key words: GLUT4, muscle contraction, VAMP, translocation.