A family of accessory beta subunits significantly contributes to the functional diversity of large-conductance, Ca(2+)- and voltage-dependent potassium (BK) channels in native cells. Here we describe the functional properties of one variant of the beta subunit family, which confers properties on BK channels totally unlike any that have as yet been observed. Coexpression of this subunit (termed beta3) with Slo alpha subunits results in rectifying outward currents and, at more positive potentials, rapidly inactivating ( approximately 1 msec) currents. The underlying rapid inactivation process results in an increase in the apparent activation rate of macroscopic currents, which is coupled with a shift in the activation range of the currents at low Ca(2+). As a consequence, the currents exhibit more rapid activation at low Ca(2+) relative to any other BK channel subunit combinations that have been examined. In part because of the rapid inactivation process, single channel openings are exceedingly brief. Although variance analysis suggests a conductance in excess of 160 pS, fully resolved single channel openings are not observed. The inactivation process results from a cytosolic N-terminal domain of the beta3 subunit, whereas an extended C-terminal domain does not participate in the inactivation process. Thus, the beta3 subunit appears to use a rapid inactivation mechanism to produce a current with a relatively rapid apparent activation time course at low Ca(2+). The beta3 subunit is a compelling example of how the beta subunit family can finely tune the gating properties of Ca(2+)- and voltage-dependent BK channels.