PDGF stimulation of skeletal muscle, cultured myotubes, and 3T3L1 adipocytes result in Glut4 translocation, albeit to a reduced level compared to insulin. To address the mechanism of PDGF action, we have determined that the Syntaxin 4 negative regulatory protein, Munc18c undergoes PDGF stimulated phosphorylation on tyrosine residue 521. The tyrosine phosphorylation of Munc18c on Y521 occurred concomitant with the dissociation of the Munc18c protein from Syntaxin 4 in a time frame consistent with Glut4 translocation. Moreover, expression of the wild type Munc18c protein did not inhibit PDGF-induced Glut4 translocation whereas expression of Y521A-Munc18c mutant was inhibitory and failed to dissociate from Syntaxin 4. In contrast, expression of either wild type Munc18c or the Y521A-Munc18c mutant both resulted in a marked inhibition of insulin-stimulated Glut4 translocation. Taken together these data demonstrate that one mechanism accounting for the PDGF induction of Glut4 translocation is the suppression of the Munc18c negative regulation of Syntaxin 4 function.