1. Recordings of the activity of the large conductance Ca2+-activated K+ (BK) channel from over 90 % of inside-out patches excised from acutely dissociated hippocampal CA1 neurones revealed an inactivation process dependent upon the presence of at least 1 microM intracellular Ca2+. Inactivation was characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consisted of very short openings (time constant (tau), approximately 0.14 ms) and rare longer duration openings (tau, approximately 3.0 ms). 2. Channel inactivation occurred with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside-out recording. Inactivation persisted whilst recording conditions were constant. 3. Inactivation was reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation was also reversed when the intracellular Ca2+ concentration was lowered to 100 nM and was permanently removed by application of trypsin to the inner patch surface. In addition, inactivation was perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. 4. During inactivation, channel Po was greater at hyperpolarized rather than depolarized potentials, which was partly the result of a greater number of longer duration openings. Depolarizing voltage steps (-40 to +40 mV) applied during longer duration openings produced only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (tau, approximately 3.8 ms). 5. These data suggest that hippocampal BK channels exhibit a Ca2+-dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation was removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking particle may act at the intracellular side of the channel.