The effects of membrane surface charge originated from lipid head groups on ion channels were tested by analyzing the activity of single large conductance Ca2⁢-activated K⁢ (maxi K) channel from rat skeletal muscle. The conductances and open-state probability (Po) of single maxi K channels were compared in three types of planar lipid bilayers formed from a neutral phosphatidylethanolamine (PE) or two negatively-charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). Under symmetrical KCl concentrations (3∼1,000 mM), single channel conductances of maxi K channels in charged membranes were 1.1∼1.7 times larger than those in PE membranes, and the differences were more pronounced at the lower ionic strength. The average slope conductances at 100 mM KCl were 251⁑9.9, 360⁑8.7 and 356⁑12.4 (mean⁑SEM) pS in PE, PS and PI membranes respectively. The potentials at which Po was 1/2, appeared to have shifted left by 40 mV along voltage axis in the membranes formed with PS or PI. Such shift was consistently seen at pCa 5, 4.5, 4 and 3.5. Estimation of the effect of surface charge from these data indicated that maxi K channels sensed the surface potentials at a distance of 8∼9 Å from the membrane surface. In addition, similar insulation distance (7∼9 Å) of channel mouth from the bilayer surface charge was predicted by a 3-barrier-2-site model of energy profile for the permeation of K⁢ ions. In conclusion, despite the differences in structure and fluidity of phospholipids in bilayers, the activities of maxi K channels in two charged membranes composed of PS or PI were strikingly similar and larger than those in bilayers of PE. These results suggest that the enhancement of conductance and Po of maxi channels is mostly due to negative charges in the phospholipid head groups.