Streaming potentials arising across large-conductance Ca2+-activated K+ channels incorporated into planar lipid bilayers were measured. Ca2+-activated channels obtained either from skeletal muscle or from smooth muscle membranes were used. Streaming potentials were extracted from the current-voltage relationship for the open channel obtained in the presence of an osmotic gradient. The osmotic gradient was established by adding glucose to one side of the membrane. At 300 mM KCl, the average streaming potential was 0.72 mV/osmol per kg for t-tubule channels and 0.83 mV/osmol per kg for smooth muscle channels. Streaming potential values depend on KCl concentration, they decrease as KCl concentration increases, and the value obtained by extrapolation to zero KCl concentration is 0.85 mV/osmol per kg. Assuming that water and ions cannot pass each other, at least in a region of the channel, the streaming potential values obtained indicate that this region contains a minimum of two and a maximum of four water molecules. It is concluded that the channel has a narrow region with a length of 0.6-1.2 nm.