Liposome permeability to net proton-hydroxide flux was compared under two conditions that have produced extreme values in previous studies. In the first [Nichols, J.W. & Deamer, D.W. (1980) Proc. Natl. Acad. Sci. USA 77, 2038-2042] small pH gradients were permitted to decay near pH 7 and permeability coefficients in the range of 10(-4) cm/sec were obtained. In the second [Nozaki, Y. & Tanford, C. (1981) Proc. Natl. Acad. Sci. USA 78, 4324-4328] the decay rate of large pH gradients (approximately equal to 3 pH units) was measured and apparent permeability coefficients in the range of 10(-9) cm/sec were reported. We have repeated the conditions of the latter study and have found that the decay of large pH gradients produces a diffusion potential that limits net proton-hydroxide flux. The permeability coefficients calculated from the flux are therefore underestimated and do not reflect intrinsic proton permeability. With the same liposome preparations under conditions in which small gradients decay so that diffusion potentials are negligible, the measured permeability coefficient is near 10(-4) cm/sec, as reported earlier.