Single channel recording techniques were applied to the study of activation and inactivation of Ca2+-activated K+ channels in excised patches of membrane from rat muscle grown in culture. The concentration of intracellular surface Ca2+ was 0.6 microM in all experiments. The time course of the averaged open probability during depolarizing voltage steps of 1 s duration was biphasic for steps more positive than 20 mV; a rapid activation phase was followed by a much slower apparent inactivation with a single exponential time constant in the range of 400-800 ms. The peak open probability and degree of inactivation increased as the steps were made more positive (+30 to +80 mV) or the holding potential more negative (+30 to -40 mV). A conditional probability analysis of the open intervals immediately adjacent to the long-duration shut intervals resulting from inactivation revealed that transitions to and from the inactivated state occurred almost exclusively via a short-duration open state (mean lifetime less than 200 microseconds). The rate of transition from the short-duration open state to the inactivated state was rapid (typical rate constant 1879/s) and was sufficiently probable that as many as one out of every three short-duration openings were followed by inactivation. Normal (non-inactivating) closure of the channel from this open state was also rapid (rate constant 4003/s). At constant voltage (+50 mV) and Ca2+ (0.6 microM), the channel opened to the short-duration open state approximately every 75 ms, suggesting that the slow inactivation of the averaged open probability might have been limited in part by the rate at which the channel entered the short-duration open state.