The purpose of this study is to delineate the immediate sources and fractional turnover of high density lipoprotein (HDL) esterified cholesterol in man. Various labeled preparations were administered in 11 experiments to six subjects who had either a complete bile fistula (maximally stimulated cholesterol metabolism) or an intact enterohepatic circulation. The administered tracers included [(3)H]mevalonic acid; [(14)C]cholesterol bound to albumin; low density lipoprotein (LDL) free [(3)H] or [(14)C]cholesterol; HDL free [(3)H] or [(14)C]cholesterol; HDL esterified [(3)H]cholesterol; and LDL esterified [(3)H]cholesterol. Blood samples were obtained at frequent intervals for up to 5 d after the administration of tracers. The mass and radioactivity in individual plasma lipoprotein (very low density lipoprotein [VLDL], HDL, and LDL) free and esterified cholesterol were determined. The data were subjected to multicompartmental analysis using the SAAM-27 computer program. The analysis revealed that plasma free cholesterol was not the only immediate source of either a single- or two-compartment HDL ester system. When LDL esters and plasma (HDL) free cholesterol were tested together as sources of one HDL ester compartment, data from all the experiments were readily fit. The fluxes arrived at with the final model indicated that only approximately 20% of the esterified cholesterol in HDL was newly synthesized from plasma (HDL) free cholesterol (2.36 mumol/min); the remaining 80% was from LDL ester (8.92 mumol/min). The presence of a bile fistula had no obvious effect on HDL esterified cholesterol metabolism. The rate of HDL cholesterol ester turnover was 3-12 times/d, indicating that the ester component of the HDL particle is in a very dynamic state.