Most BK-type voltage- and Ca2+-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore- blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 μM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.