Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca(2+) at a hair cell's operating voltage range (spanning -40 to -60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca(2+) concentrations at -50 mV. We have cloned the chick β(4)- and β(1)-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The β(4)-subunit in particular is responsible for the BK channel's increased responsiveness to Ca(2+) at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both β-subunits suggest additional mechanisms responsible for BK channel function in hair cells.