The passive proton permeability (Pnet) of apical membrane vesicles from rabbit parietal cells (gastric) and duodenal and renal cortical brush-border membranes (BBM) was determined by acridine orange fluorescence quenching. Values of Pnet were found to be gastric (4 x 10(-4) cm/s) less than duodenal (10(-3) cm/s) much less than renal (10(-2) cm/s). Arrhenius plots of the temperature profile of proton permeation of gastric vesicles was linear, whereas that of duodenal BBM displayed a discontinuity at 30-33 degrees C. Alcohols (octyl, benzyl, ethyl) increased Pnet in a concentration-dependent manner, with efficacy related to their oil-water partition coefficients. In a parallel series of experiments, structural parameters of the vesicle membrane lipids (fluidity) were monitored from both the steady-state and time-resolved fluorescence anisotropy of diphenylhexatriene. Fluidity of the membranes was unrelated to Pnet (renal congruent to duodenal less than gastric). Gastric vesicles demonstrated a linear Arrhenius plot of temperature dependence for fluidity, whereas duodenal BBM demonstrated a discontinuity. Membrane fluidity of gastric and duodenal vesicles was increased by alcohols, with the same potency as for Pnet, and these two variables were significantly correlated after perturbation with alcohols. Thus the fluidity of the lipid bilayer is not the major determinant of Pnet, but alteration of its structural parameters, as reflected by fluidity, produces parallel changes in Pnet.