Syntaxin 1A (Syn1A) and Munc18a play essential roles in vesicular trafficking and exocytosis. The molecular mechanism underlying the sorting of these two proteins to their physiological sites of action remains poorly understood. Here the localization of syntaxin1A (Syn1A) and Munc18a was analyzed in baby hamster kidney (BHK-21) cells and human embryonic kidney (HEK293) cells. The rat Syn1A gene was fused to the gene encoding the enhanced green fluorescent protein (EGFP). Munc18a was labeled with the red fluorescence protein (TDimer2) at its C terminal. The proteins were expressed by transient transfection in either BHK-21 or HEK293 cells. Under fluorescence microscopy, it was shown that Syn1A was shown to be transported to the plasma membrane. While Munc18a exhibited mainly cytosolic distribution when expressed alone. However, upon coexpression with Syn1A, Munc18a is translocated to the plasma membrane. In addition, a N-terminal truncated mutant Syn1A failed to localize at the plasma membrane, suggesting that the cytoplasmic domain of Syn1A is important for its sorting and localization.