The rate of H⁺ transport in collecting duct cells is regulated by exocytic insertion of H⁺-ATPase-laden vesicles into the apical membrane. The SNARE proteins (syntaxin 1A, SNAP-23 and VAMP) are critical in this exocytic process. Syntaxin 1A, like other isoforms of syntaxin, contains 3 main domains, SNARE N (amino terminal residues 1-160), H3 (residues 161-264) and carboxyl-terminal transmembrane domain (residues 265-288). We have shown previously that although several syntaxin isoforms form SNARE fusion complexes through the H3 domain, only syntaxin 1A, through its H3 domain, also binds H⁺-ATPase. This finding raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H⁺-ATPase and for SNARE proteins. To investigate this hypothesis, a series of truncations in the H3 domain and some chimeric molecules of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined by pull down assay the amount of H⁺-ATPase and SNARE proteins, SNAP23 and VAMP, in rat kidney medullary homogenate that complexed with GST-syntaxin molecules attached to Sepharose-glutathione beads. Full-length syntaxin isoforms and syntaxin-1AC (AA1-264) formed complexes with H⁺-ATPase and SNARE proteins, SNAP23 and VAMP. Utilizing truncation experiments, a cassette within the H3 portion was found that bound H⁺-ATPase (AA 235-264) and another that bound SNAP23 and VAMP (AA 190-234) to an equivalent degree as full-length synatxin. However, AA 235-264 cassette alone without the SNARE N (AA 1-160) does not bind, but requires ligation to the SNARE N to bind H⁺-ATPase. When this latter chimeric construct was transfected into the IMCD cells it interacted with H⁺-ATPase and inhibited the Na⁺ independent pHi recovery, an index of H⁺ -ATPase mediated proton secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding and targeting of the H⁺-ATPase to the apical membrane and may confer specificity of syntaxin 1A in the process of H⁺-ATPase exocytosis.