Stimulation of VLDL production by increasing fatty acid availability is now well established. However, a possible regulatory role of glycerol, another lipid precursor, in VLDL synthesis by the liver has not yet been substaniated. The present experiments investigate this problem using the isolated perfused rat liver. [14C] Glycerol uptake and metabolism were studied at two different glycerol concentrations: 1 mumol/perfusate (control) or 1.6 mmol/perfusate. VLDL production and lipid synthesis were investigated using [14C]leucine and several labelled fatty acids as precursors in control and glycerol-overloaded livers. Neoglycogenesis and lipogenesis from glycerol carbons are negligible in our conditions. The absolute amount of glycerol, but not the precentage, taken up by the liver, increased after raising its concentration in the perfusate. A major part of exogenous (plasmatic) glycerol was esterified with endogenous (non plasmatic) fatty acids. Incorporation of radioactive fatty acids into liver or plasma lipids was lower than in the the control group. Significant differences were observed between saturated and unsaturated fatty acids used as lipid precursors. Production of VLDL as assessed by radioactive leucine and fatty acid incorporation in the VLDL of the perfusate was depressed by glycerol. Glycerol partly inhibits the normal stimulation of VLDL production by plasmatic fatty acid overload.