The purpose of this study was to quantitate the contribution of newly synthesized cholesterol to bile and plasma in humans. Eight healthy volunteers were intravenously infused with 0.125 mmol of [1-(13)C]acetate per kilogram per hour for 15 h. During continuous enteral nutrition, plasma aliquots and samples of duodenal bile were collected hourly. The trimethysilylether of unesterified cholesterol was analyzed by gas chromatography-mass spectrometry for quantitation of the mass fragments M(+0) [mass-to-charge ratio (m/z) 368], M(+1) (m/z 369), M(+2) (m/z 370), M(+3) (m/z 371), and M(+4) (m/z 372). The fractional syntheses of plasma and biliary cholesterol were determined using mass isotopomer distribution analysis. After 6 h of infusion, the 13C enrichment of the acetate pool remained constant at 12%. The fractional synthesis increased continuously during [13C]acetate infusion and reached 4.2% and 5.3% in cholesterol of plasma and bile, respectively. Both were highly correlated, but the fractional synthesis of biliary cholesterol exceeded that of plasma (P < 0.05). It may be concluded that the contribution of de novo cholesterol synthesis to bile exceeds that to plasma but is minor in humans.