A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K+ concentration that could not be described by simple Michaelis-Menten saturation. At low K+ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4 m KCl. At the same Ca2+ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K+ and Ca2+ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K+-conduction and Ca2+-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 A from the surface.