To evaluate the possible role of a putative vesicle-targeting protein, syntaxin-4, in vasopressin-regulated trafficking of aquaporin-2 water channel vesicles to the apical plasma membrane of renal collecting duct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney. Immunochemical studies used an affinity-purified, peptide-directed polyclonal antibody to rat syntaxin-4. Immunoblots using membrane fractions from inner medullary collecting duct (IMCD) cell suspensions revealed a solitary protein of 36 kD, the expected molecular mass of syntaxin-4. This protein was enriched in a plasma membrane-enriched membrane fraction from IMCD cells. Immunoperoxidase immunocytochemistry in 0.85-microm cryosections from rat inner medulla revealed discrete labeling of the apical plasma membrane of IMCD cells. RT-PCR demonstrated the presence of syntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct sequencing of the PCR product. In addition, RT-PCR experiments demonstrated syntaxin-4 mRNA in glomeruli, vasa recta, connecting tubules, and thin descending limbs of Henle's loops. The demonstrated localization of syntaxin-4 in the apical plasma membrane of collecting duct principal cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports the view that these proteins could play a role of aquaporin-2 vesicle targeting to the apical plasma membrane.