We tested the effects of membrane phospholipids on the function of high-conductance, Ca²⁺-activated K⁺ channels from the basolateral cell membrane of rabbit distal colon epithelium by reconstituting these channels into planar bilayers consisting of different 1:1 mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambient K⁺ concentrations single-channel conductance is higher in PE/PS and PE/PI bilayers than in PE/PC bilayers. At high K⁺ concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayers increases channel conductance, whereas the positively charged dodecyltrimethylammonium has the opposite effect. All these findings are consistent with modulation of channel current by the charge of the lipid membrane surrounding the channel. But the K⁺ that permeates the channel senses only a small fraction of the full membrane surface potential of the charged phospholipid bilayers, equivalent to separation of the conduction pathway from the charged phospholipid head groups by 20 Å. This distance appears to insulate the channel entrance from the bilayer surface potential, suggesting large dimensions of the channel-forming protein. In addition, in PE/PC and PE/PI bilayers, but not in PE/PS bilayers, the open-state probability of the channel decreases with time ("channel rundown"), indicating that phospholipid properties other than surface charge are required to maintain channel fluctuations.