We have investigated the conductance properties of large-conductance Ca2+-activated K+ (BKCa) channels formed by stable expression of the rSlo gene in HEK 293 cells. Single-channel recordings were obtained from inside-out patches excised into solution containing 100 microM Ca2+ to ensure a relatively high open probability over the range of membrane potentials studied (-120 to +100 mV). The unitary conductance of these channels at +80 mV was 221.6+/-5.4 pS in symmetrical 140 mM K+. Decreasing the K+ concentration on either side of the membrane, while maintaining ionic strength by adding N-methyl d-glucamine (NMDG+), reduced the unitary conductance. The reduction in conductance was greater when internal K+ was lowered by replacement with NMDG+. However, if sucrose was used as the internal K+ substitute instead of NMDG+ the reduction in unitary conductance was similar to that seen on reducing external K+. A rate-theory model whereby NMDG+ produces a very rapid block of the BKCa channel from the inside, but not the outside, is able to describe our results.