The present study investigated the effect of serum lipoproteins on sterol synthesis by isolated rat hepatocytes. These cells were maintained in culture medium for 24 hr and incubated for the same period of time with increasing concentrations of serum lipoproteins (5-150 microgram of lipoprotein-protein per ml) isolated from different animal species. The viability of the cells was ascertained by their ability to synthesize cholesterol and protein and to secrete serum proteins into the medium. Rat VLDL and LDL did not alter sterol synthesis, which was stimulated instead by HDL. Rat serum chylomicrons were also ineffective. Human LDL significantly reduced the synthesis of sterols from both acetate and tritiated water; this effect was also induced by human VLDL to a reduced extent. VLDL isolated from hypercholesterolemic rabbit (VLDLC) strongly inhibited sterol synthesis from acetate but not from mevalonate. Cholesteryl-ester-rich VLDL isolated from a patient with type III hyperlipidemia (type III VLDL) were more effective than normal VLDL in suppressing sterol synthesis from acetate. The implications of these findings are discussed with regard to the possible role of cholesteryl-ester-rich lipoproteins on the in vivo regulation of sterol synthesis in the liver.