The large conductance calcium-activated potassium channel, or BK_Ca channel, plays an important feedback role in a variety of physiological processes including neurotransmitter release and smooth muscle contraction. A number of reports have suggested that this channel forms a stable complex with regulators of its function, including several kinases and phosphatases . To further define such signaling complexes, we used the yeast-two-hybrid system to screen a human aorta cDNA library for proteins that bind to the BK_Ca channels intracellular, C-terminal tail. One of the interactors we identified is the protein RACK1. RACK1 is a member of the WD40 protein family, which also includes the G-protein subunits. Consistent with an important role in BK_Ca-channel regulation, RACK1 has been shown to be a scaffolding protein that interacts with a wide variety of signaling molecules including cSRC and PKC. We have confirmed the interaction between RACK1 and the BK_Ca channel biochemically with GST pull down and co-immunoprecipitation experiments. We have observed some co-localization of RACK1 with the BK_Ca channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BK_Ca channels biophysical properties. Thus, RACK1 binds to the BK_Ca channel and it may form part of a BKCa-channel regulatory complex in vascular smooth muscle.