Like other local anesthetics, bupivacaine exerts its clinical effects through a use- and state-dependent block of voltage-dependent sodium channels. Here we report that bupivacaine also targets the calcium-activated large-conductance potassium (BK or MaxiK) channel. In whole-cell recordings from HEK293 cells transiently expressing MaxiK a-subunits, bupivacaine (30mM-1mM) produced a dose-dependent inhibition of MaxiK currents. The Hill plot of normalized dose-response data revealed half-maximal inhibition (IC₅₀) at 197±12 mM (n = 6). The Hill coefficient was close to unity (1.5) indicating little cooperativity in the inhibition of MaxiK channels by bupivacaine. Suppression of MaxiK currents was associated with a shift of the voltage dependence of current activation to more positive potentials, as expected for a local anesthetic. In single-channel recordings from inside-out patches, increasing concentrations of bupivacaine caused a progressive "flicker block" of the channel that precluded unambiguous resolution of the conducting state owing to the ultrafast gating kinetics. To explore the site of bupivacaine action, we compared its blocking effect with that of U73122. We recently identified U73122 as a novel and potent blocker of MaxiK channels most likely interacting with its long C-terminus to suppress channel openings. U73122 slowed the gating kinetics of MaxiK channels dramatically. When we then added bupivacaine, the remaining channel activity was still subjected to flicker block. Vice versa, when bupivacaine-treated MaxiK channels were exposed to U3122, channel activity was strongly reduced, but the flicker block itself appeared unaffected. In view of the qualitatively different and additive effects of the two blockers, they would be expected to act at independent sites of the channel protein. Our data would be consistent with the notion that U73122 binds to a region outside the pore, whereas bupivacaine is likely to occupy a site in the pore region of MaxiK.