Pantophysin, a protein related to the neuroendocrine-specific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [(32)P]orthophosphate-labeled cells, pantophysin was phosphorylated in the basal state. This phosphorylation was unchanged in response to insulin; however, insulin stimulated the phosphorylation of a 77-kDa protein associated with alpha-pantophysin immunoprecipitates. Although the functional role of pantophysin in vesicle trafficking is unclear, its presence on GLUT4 vesicles is consistent with the emerging role of soluble N-ethylmaleimide-sensitive protein receptor (SNARE) factor complex and related proteins in regulated vesicle transport in adipocytes. In addition, pantophysin may provide a marker for the analysis of other vesicles in adipocytes.