Using the planar lipid bilayer method, we investigated the effect of d-tubocurarine (dTC) on the extracellular side of large-conductance Ca2+-activated K+ channel from rat brain. When the initial open probability (Po) of the channel was relatively high, dTC decreased channel activity in a concentration dependent manner. In contrast, when the initial Po was lower, sub-micro molar dTC increased channel activity by destabilizing the closed states of the channel. Further addition of dTC up to micro molar range decreased channel activity. This dual effect of dTC implicates that there exist at least two different binding sites for dTC.