We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that, although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.Branching morphogenesis is a developmental process involved in the formation of many organs, including mammary gland, lung, kidney, and salivary gland. In the mammary gland, branching morphogenesis allows the primitive anlage to develop into the highly ramified mammary ductal tree. Investigations of the signaling processes involved in mammary ductal branching have revealed that branching requires the presence of a growth factor and the morphogen epimorphin (1). Epimorphin binds to mammary epithelial cells through ␣v-integrins (2), activating morphogenic processes that are dependent upon the transcription factor CCAAT/enhancer-binding protein- (C/EBP) 5 (3), and mediating branching and invasion into the extracellular matrix through activation of matrix metalloproteinase-3 (MMP3) (4). Although epimorphin is required for branching morphogenic processes, overexpression of epimorphin can lead to pathological consequences, including ductal hyperplasia and mammary cancer (3, 5). Epimorphin plays a role in morphogenesis of other epithelial organs as well, including kidney (6), pancreas (7,8), hair (9), intestine (10, 11), and lung (12).Although epimorphin was first identified as an extracellular morphogen through the use of function-blocking antibodies in lung and skin organ culture assays (12, 13), the same molecule was later found to function in the cytoplasm as syntaxin-2, a member of the syntaxin family of proteins that controls vesicle fusion (14, 15). However, the idea that epimorphin/syntaxin-2 might have distinct roles dependent upon its location on the outside or inside of the plasma membrane is controversial (14 -16), in part because epimorphin lacks a canonical peptide signal sequence to direct extracellular localization through the ER/Golgi path...