High conductance calcium-activated (maxi-K) channels are potently blocked by a family of indole diterpenes that includes paxilline. Paxilline stimulates binding of charybdotoxin (ChTX) to maxi-K channels in vascular smooth muscle and blocks these channels in electrophysiological experiments (Knaus et al., 1994b). These results suggested that paxilline blocked maxi-K channels at a site distance from the ChTX binding site located near the external entrance to the pore. Here we have examined block of the cloned alpha subunit (slo) of the maxi-K channel in excised membrane patches after internal application of paxilline. Paxilline caused a reversible inhibition of channel currents with slow washout kinetics. In the presence of 10 muM intracellular calcium, paxilline blocked currents elicited by brief voltage pulses with a Ki of 1.9 nM and a Hill coefficient near one. Changing the internal calcium by the fold caused a two to three fold change in the Ki for paxilline block, with less block occurring at high calcium concentrations. Paxilline reduced the maximum of the conductance-voltage relation in a calcium-sensitive manner with less block occurring at high calcium concentrations, and caused a 20 mV depolarizing shift in the midpoint for channel opening. The time-course of relief of paxilline block by elevated calcium was more rapid than washout of paxilline suggesting an allosteric interaction between calcium and paxilline.