The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.