Large conductance, calcium-activated K+ channels (BKCa channels) are regulated by several distinct mechanisms, including phosphorylation/dephosphorylation events and protein-protein interactions. In this study, we have examined the interaction of BKCa channels with syntaxin 1A, a SNARE protein that is reported to modulate the activity and/or localization of different classes of ion channels. Using a reciprocal co-immunoprecipitation strategy, we observed that native BKCa channels in rat hippocampus co-associate with syntaxin 1A, but not the closely related homologue syntaxin 3. This BKCa channel- syntaxin 1A interaction could be further demonstrated in a non-neuronal cell line (HEK 293 cells) following co- expression of rat syntaxin 1A and BKCa channels cloned from either mouse brain or bovine aorta; however, co- expression of these same channels with syntaxin 3 did not lead to a detectable protein-protein interaction. Immunofluorescent co-staining of HEK 293 cells expressing BKCa channels and syntaxin 1A demonstrated overlapping distribution of these two proteins in situ. Functionally, co-expression of BKCa channels with syntaxin 1A, but not syntaxin 3, was observed to enhance channel gating and kinetics at low concentrations (1-4 & [mu]M) of free cytosolic calcium, but not at higher concentrations (10-100 µM), as judged by macroscopic current recordings in excised membrane patches. Interactions of BKCa channels with neighboring membrane proteins may thus play important roles in regulating the activity and/or distribution of these channels within specialized cellular compartments.