High-density lipoprotein (HDL) participates in the transfer of cholesterol to the liver, in which it is subsequently excreted into bile as bile acid and cholesterol. In this study, the effect of essential fatty-acid (EFA) deficiency on cholesterol contribution from HDL subfractions to bile was investigated. Rats that were rendered EFA-deficient over 4 weeks displayed changes in their plasma HDL subfractions and liver tissue fatty acids. Plasma linoleic (18:2n6), linolenic (18:3n3,) and arachidonic (20:4n6) acids decreased, whereas palmitoleic (16:1n7) and eicosatrienoic (20:3n9) acids increased. EFA deficiency was confirmed by an elevation of the 20:3(n-9)/20:4(n-6) index. To examine the hepatic handling of lipoprotein-derived cholesterol, HDL2 and HDL3 from donor rats were isolated, labeled with [14C]-cholesterol, and injected iv into EFA-deficient and normal rats with a bile fistula. In HDL subfractions from control rats, no significant variations were noted in the specific activity of cholesterol output in both groups of EFA recipient rats; however, the output of biliary bile acids was significantly decreased in EFA-deficient rats following the administration of labeled HDL3. In HDL2 and HDL3 originating from EFA-deficient rats, a decrease in the specific activity of both biliary cholesterol and bile acid output was recorded in EFA-deficient rats. Concomitant with the defective HDL2- and HDL3-[14C] cholesterol translocation into bile of EFA-deficient rats, increased hepatic very-low-density lipoprotein (VLDL)-[14C] cholesterol secretion was observed in vivo. HDL2 and HDL3 particles, derived from EFA-deficient rats, had an altered composition including a depletion in apo A-I and an enrichment in apo E isoforms, which are the the two major HDL apolipoproteins involved in the delivery of cholesterol to the liver. Taken together, these results show that normal EFA status is necessary for efficient HDL-cholesterol processing by the liver.