The ability of high-density lipoprotein (HDL) to reduce the cholesterol content was studied in cultured fibroblasts enriched with cholesterol esters. Incubation of cholesterol-enriched cells with HDL in a final concentration of 1 g protein/l for 24 h reduced the total and esterified cholesterol content by 23% as compared with control fibroblasts incubated with albumin. Similar cholesterol efflux was obtained with HDL isolated from lecithin:cholesterol acyltransferase (LCAT)-deficient plasma. The HDL3 subfraction isolated by rate-zonal ultracentrifugation contained the major part of the cholesterol-depleting effect. HDL or HDL3 decreased CoA:cholesterol acyltransferase (ACAT) activity to 5% of the level found in control fibroblasts within 8 h of incubation. These findings suggest that ACAT activity is sensitive to a pool of intracellular cholesterol, which can be mobilized by the addition of HDL to the culture medium, and that ACAT activity is a useful measure of cholesterol efflux from cultured fibroblasts.