Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250-300 pS and 9-14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at-50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at -50 mV, and its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.