The patch-clamp technique was used to study the effect of intracellularly added inactivating "ball" peptide (BP) of the Shaker B K+ channel upon Ca(2+)-dependent inwardly rectifying K+ channels of the intermediate conductance type expressed in HeLa cells. Intracellular BP caused only moderate inhibition of outward K+ currents when assayed at an intracellular Ca2+ concentration of 100 nmol/l. Increasing intracellular Ca2+ levels led in itself to some voltage-dependent blockade of K+ currents, which was absent when high extracellular K+ was used. An additional strong blockade by intracellular BP was nevertheless observed both in Na(+)- and K(+)-rich extracellular solutions. A non-inactivating BP analogue had no effect. At this higher intracellular Ca2+ concentration the inhibition of these intermediate conductance Ca(2+)-dependent channels by BP was voltage-dependent, being absent at hyperpolarizing potentials, and could be relieved by increasing extracellular K+. These data suggest that BP acts at an internal pore site in Ca(2+)-dependent intermediate conductance K+ channels of HeLa cells, and that these might possess a receptor site for the peptide similar to that of other K+ channels such as Ca(2+)-activated maxi-K+ channels.