Abstract Livers from normal fed male rats were perfused in vitro with equimolar quantities of either palmitic acid or oleic acids, and, after an experimental period of 4 hours, the very low density lipoprotein (VLDL) was isolated from the perfusate by ultracentrifugation in zonal rotors. The VLDL was characterized by rate-zonal mobility, by lipid composition, and by electrophoretic mobility on paper. Approximately 50% more VLDL triglyceride was secreted by the liver when oleate was the substrate than when palmitate was the substrate. The VLDL produced when palmitate was infused was a more dense particle and had a slower rate-zonal mobility under our conditions of zonal ultracentrifugation than did the VLDL produced from oleate. When palmitate was the substrate, the VLDL secreted by the liver was characterized by a single, apparently homogeneous peak in the density gradient in the zonal rotor, whereas the VLDL produced from oleate appeared to consist of at least two components; the lighter component behaved as a particle in its rate-zonal mobility, whereas the heavier component was similar to the VLDL produced from palmitate. Both fractions derived from oleate and the lipoprotein derived from palmitate migrated on paper as a VLDL (pre-β-lipoprotein); the lipid composition of the chylomicron-like VLDL secreted by the liver after infusion of oleate was similar to that of the chylomicrons isolated from the perfusate at the beginning of the experiment although the chylomicrons remained at the origin and did not migrate as a VLDL on paper electrophoresis. The less dense VLDL produced from oleate, furthermore, had a lipid composition which differed from that of the VLDL produced from palmitate; the lighter VLDL produced from oleate contained half as much cholesterol and phospholipid per µmole of triglyceride (TG) than either the VLDL produced from palmitate or the heavier VLDL secreted after infusion of oleate. Even though more triglyceride was secreted per gram of liver when oleate was the substrate than when palmitate was infused, the total quantity of cholesterol and phospholipid secreted, respectively, in response to either fatty acid was the same, e.g. twice as much cholesterol and phospholipid was secreted per g of liver per µmole of VLDL triglyceride derived from palmitate than from the lighter lipoprotein produced from oleate. We can conclude from these data that certain physical properties and the lipid composition of the VLDL secreted by the liver were prescribed by infusion of either palmitate or oleate; it remains to be determined whether these observations can be extended to some general relationship describing effects of saturated and unsaturated free fatty acid (FFA) on the hepatic production and properties of the VLDL. The concentration of TG, phospholipid, and cholesterol in the VLDL of serum in vivo, which is the result, in part, of hepatic secretion of VLDL, must also be regulated by the chemical structure of the FFA from which the triglyceride fatty acids are derived.