The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alpha SNAP as an interactive partner of G alpha(12) using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of alpha SNAP with G alpha(12) in COS- 7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha(12) ( 1-37 amino acids) was necessary and sufficient for its interaction with alpha SNAP. G alpha(13) with its N-terminal extension replaced by that of G alpha(12) acquired the ability to bind to alpha SNAP, whereas G alpha(12) with its N terminus replaced by that of G alpha(13) lost this ability. Using four point mutants of alpha SNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alpha SNAP was involved in its interaction with G alpha(12). Co-transfection of human umbilical vein endothelial cells with G alpha(12) and alpha SNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alpha SNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha 12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G(12) and alpha SNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.