ROMK potassium channels are present in cortical collecting duct (CCD) of kidney and serve as apical exit pathways for K⁺ secretion in this nephron segment. K⁺ secretion in the CCD is regulated by multiple factors. In this study, we show that syntaxin 1A, but not syntaxin 3 or 4, inhibited whole-cell ROMK currents in Xenopus oocytes. Syntaxin 1A, but not syntaxin 3 or 4, interacted with the C-terminal cytoplasmic domain of ROMK in intro. Co-expression with synaptobrevin 2 reversed inhibition of whole-cell ROMK currents by syntaxin 1A. In excised inside-out membranes of oocytes, application of fusion proteins containing cytoplasmic region of syntaxin 1A to cytoplasmic face caused a dose-dependent inhibition of ROMK. We further examined regulation of the K⁺ channels in the CCD by syntaxin 1A. Application of botulinum toxin C1 to the excised inside-out membranes of CCD caused an increase in the activity of K⁺ channels. In contrast, application of toxin B had no effects. These results suggest that syntaxin 1A causes a tonic inhibition of the K⁺ channels in the apical membrane of CCD. Binding of synaptobrevin 2 to syntaxin 1A during docking and fusion of transport vesicles to the plasma membranes of CCD may lead to activation of these channels.