Nonspecific lipid transfer protein accelerated cholesterol exchange from brush border vesicles according to a biphasic time course, but sonicated vesicles made from brush border phospholipids and glycosphingolipids showed a single phase exchange. Removal of surface protein with papain or opening brush border vesicles with deoxycholate did not abolish the biphasic exchange pattern. In brush border vesicles treated with cholesterol oxidase, 21 +/- 10% of the free cholesterol was oxidized rapidly, and the remaining cholesterol was oxidized at a slower rate. Opening vesicles with sodium deoxycholate or treatment with phospholipase C, which degraded 55% of the phospholipids, did not increase the size of the rapidly oxidizable cholesterol pool. The rapidly exchangeable and the rapidly oxidizable cholesterol pools appear to represent the same fraction. In double-labeled brush border vesicles 27 +/- 9% of the cholesterol is present in a readily accessible pool, which slowly equilibrates with the remaining membrane cholesterol. The fractional turnover rate of cholesterol in the readily accessible pool equals 0.07 +/- 0.04 h-1 and is increased to 3.35 h-2 by 12 micrograms/ml of nonspecific lipid transfer protein. The heterogeneous distribution of cholesterol in the intact brush border vesicles may not reflect an inside-outside distribution or interaction of cholesterol with membrane lipids but rather an association of more than two-thirds of the membrane cholesterol with a membrane protein fraction.