The effect of cholesterol on the diffusional permeability of water in suspensions of human erythrocytes was studied by means of pulsed field gradient NMR, which unlike the relaxation NMR method avoids the use of Mn2+ ions. The analysis allows the internal and external diffusion coefficients, as well as the lifetime characterizing the rate of exchange between the two regions, to be extracted from the data. The cholesterol content of the erythrocyte membranes was altered by incubating the cells with sonicated dispersions of cholesterol/dipalmitoyl phosphatidylcholine at 310 K. It was shown that decreasing the molar ratio of cholesterol to phospholipid (C/P ratio) of the membrane, from a mean value of 0.92 for normal cells (controls) to a value of 0.46, had little effect on the intracellular mean residence lifetime and the diffusional permeability. Enriching the cholesterol content of the membrane, however, had a marked effect on the exchange lifetime and the diffusional permeability. At a C/P ratio of approximately 1.5 the rate of transport was reduced approximately 3.5-fold. A further increase of the cholesterol content, to a C/P ratio of approximately 1.9, resulted in an enhancement of the rate of transport back to a normal (control) value, which was characterized by a lifetime of 8-9 ms. The combined inhibition of the water permeability by cholesterol and pCMBS for cells with C/P ratios of 1.44 and 1.54, and by pCMBS alone for cells with a control C/P ratio resulted in the same value for Pd within experimental error.