Proton/hydroxide (HI/OH-) permeability of phospholipid bilayers is several orders of magnitude higher than alkali or halide ion permeabilities at pH 7. The objective of this study was to determine the mechanism(s) of HI/OHconductance and permeability through planar phospholipid bilayer membranes. Membranes were formed from decane solutions of bacterial phosphatidylethanolamine, diphytanoyl phosphatidylcholine, or egg phosphatidylcholine plus cholesterol. At pH 7, H'/OH-conductance (GH/OH) ranged from 2 to 6 nS cm2, corresponding to H'/OH "net" permeabilities of (0.4-1.6) x 10-5 cm sec'1. GH/OH was inhibited by serum albumin (fatty acid-free), phloretin, and low pH. GH/OH was increased by chlorodecane, long-chain fatty acids, and voltages > 80 mV. Water permeability and GH/OH were not correlated. The results suggest that the Hf/OH-charge carrier (i) is primarily anionic, (ii) crosses the membrane via nonpolar pathway(s), and (iii) can be removed from the membrane by "washing" with serum albumin. The simplest explanation is that the phospholipids contain weakly acidic contaminants that act as proton carriers at neutral pH. However, at low pH or in the presence of inhibitors, a "background" GH/OH remains that may be due to other mechanisms.Proton/hydroxide (H+/OH-) permeability of phospholipid bilayers at physiological pH is several orders of magnitude higher than alkali or halide ion permeability (1-9), but the mechanism(s) of H+/OH-permeability is unknown. The primary purpose of this study was to determine the mechanism(s) of H+/OH-permeability and conductance through planar phospholipid bilayers. In order to characterize the transport mechanism(s), several inhibitors and enhancers of H+/OH-conductance (GH/OH) were identified. GH/OH was inhibited by serum albumin (fatty acid-free), phloretin, and low pH. GH/OH was increased by chlorodecane, long-chain fatty acids, and membrane voltages >80 mV. GH/OH was not affected by substituting 2H20 for H20. Water permeability was compared with H+/OH-conductance and found not to be correlated. The simplest explanation for these results is that most of the H+/OH-conductance at pH 7 is due to weakly acidic contaminants, possibly long-chain fatty acids, in the phospholipids. However, at low pH or in the presence of inhibitors, a significant "background" H+/OH-conductance remains, which may be due to other mechanisms-e.g., "water wires" (1, 2, 10, 11) or "hydrated defects" (12, 13) in the bilayer structure.METHODS AND MATERIALS Planar (Mueller-Rudin; ref. 14) membranes were formed from 2.5% (wt/vol) solutions of (i) bacterial phosphatidylethanolamine, (ii) diphytanoyl phosphatidylcholine, or (iii) egg phosphatidylcholine plus cholesterol (1: 1 molar ratio) in either n-decane or n-decane plus 1-chlorodecane (30%, vol/vol). The membranes (2.0 mm2) were formed in a symmetrical chamber with 1.1 ml of bathing solution and a small magnetic stirrer on each side of the membrane. One side of the membrane was perfused continuously to facilitate solution changes and the measurement ...