SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.