The rate of transport of protons across lipid membranes is anomalously high when compared to that of other monovalent cations. The H-bonded water-wire mechanism had been proposed earlier for explaining this anomaly. We have probed the dynamics of lipid membranes by the fluorescence probe Nile Red. Membrane composition was altered by incorporation of cholesterol. Proton transport across membranes was estimated by the rate of decay of the pH gradient monitored by pyranine fluorescence. The fluorescence lifetime and lifetime distribution (analyzed by the maximum entropy method) of Nile Red in membranes were estimated. An increase in the level of cholesterol resulted in a decrease in the rate of proton transport and increases in both the peak value and width of the lifetime distribution of Nile Red. These results are interpreted by a model wherein cholesterol causes a decrease in the water content of membranes and thereby decreases the probability of the H-bonded water wire across membranes, resulting in a decrease in proton flux.