Recycling of H(+)-ATPase to the apical plasma membrane, mediated by vesicular exocytosis and endocytosis, is an important mechanism for controlling H(+) secretion by the collecting duct. We hypothesized that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins) may be involved in the targeting of H(+)-ATPase-coated vesicles. Using a tissue culture model of collecting duct H(+) secretory cells (inner medullary collecting duct (IMCD) cells), we demonstrated that they express the proteins required for SNARE-mediated exocytosis and form SNARE-fusion complexes upon stimulation of H(+)-ATPase exocytosis. Furthermore, exocytic amplification of apical H(+)-ATPase is sensitive to clostridial toxins that cleave SNAREs and thereby inhibit secretion. Thus, SNAREs are critical for H(+)-ATPase cycling to the plasma membrane. The process in IMCD cells has a feature distinct from that of neuronal cells: the SNARE complex includes and requires the vesicular cargo (H(+)-ATPase) for targeting. Using chimeras and truncations of syntaxin 1, we demonstrated that there is a specific cassette within the syntaxin 1 H3 domain that mediates binding of the SNAREs and a second distinct H3 region that binds H(+)-ATPase. Utilizing point mutations of the B1 subunit of the H(+)-ATPase, we document that this subunit contains specific targeting information for the H(+)-ATPase itself. In addition, we found that Munc-18-2, a regulator of exocytosis, plays a multifunctional role in this system: it regulates SNARE complex formation and the affinity of syntaxin 1 for H(+)-ATPase.