Cholesterol is principally excreted from the body in bile as unesterified cholesterol (UC). Using the unesterified plant sterol, sitostanol (SIT), as a nonexchangeable analog for UC, we have found that high-density lipoproteins (HDL), but not low-density lipoproteins, provide a vehicle for the direct delivery of cholesterol to bile. To determine the mechanism for preferential cholesterol transport from HDL to bile, isolated rat livers were perfused with a reconstituted HDL, made with radiolabeled unesterified SIT, UC, and cholesteryl esters (CE). Total biliary sterol secretion was independent of the concentration of HDL added to perfusions, but with increasing HDL-SIT perfused, the proportion of SIT to cholesterol in bile was linearly increased. Biliary SIT secretion was rapid (detected within 2 to 4 minutes after reconstituted HDL was added to perfusions) and was dependent on the immediate presence of SIT in the perfusate, but independent of the amount of SIT that had accumulated in the liver. The ratio of SIT to UC was seven- to ninefold greater in bile than in the liver, consistent with preferential mobilization of membrane sterols delivered from HDL. Although radiolabeled UC as well as SIT was taken up from HDL by the liver and secreted in bile, net UC uptake could not be quantitated because of both UC exchange and a sizable enrichment of HDL with UC mass that approximated the SIT removed during the passage of HDL through the liver. These results are consistent with sterol transport to bile from HDL by a direct plasma membrane pathway and by a mechanism that appears to involve substitution of unesterified (exogenous) sterol from HDL for plasma membrane UC during transport. By this process, HDL can promote reverse cholesterol transport from peripheral tissues to bile, even without an increase in biliary cholesterol secretion.