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A glycoprotein of M(r) 160,000 (gp160) represents the major protein component of GLUT4-containing vesicles other than GLUT4 itself. These vesicles are the tissue-specific secretory-like microsomal structures that are responsible for insulin-dependent translocation of GLUT4 to the cell surface in fat and muscle cells. Our previous studies demonstrated that the expression of gp160 is also limited to fat and muscle tissues, where it is localized exclusively in GLUT4-containing vesicles, and thus, it represents a marker protein for insulin-activated glucose transport. We demonstrate here that gp160 has structural homology to aminopeptidase N and possesses aminopeptidase activity in vitro. Since aminopeptidase activity has also been found in a variety of secretory type vesicles from other tissues, it may be essential for the organization and functioning of secretory machinery in different cell types.