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Ponies fasted for up to 8 days showed, both by agarose electrophoresis and preparative ultracentrifugation, the appearance of a pre-β-migrating, very low density lipoprotein fraction in plasma. This lipoprotein differs from the very low density lipoprotein found in humans and rats in that it contains a relatively smaller amount of total cholesterol, 85% of which is present in the unesterified form. By the 8th day of fasting, plasma triglyceride concentrations had increased from a prefasting level of 20 mg/dl to as high as 1000 mg/dl. The increase in plasma lipid concentrations as a result of fasting was highly variable. Accumulation of plasma cholesterol and triglyceride after injection of Triton WR 1339 was not related to the degree of fasting hyperlipidemia. This suggests that the hyperlipoproteinemia of fasting may result from an impaired utilization of very low density lipoproteins.