The hypothesis that clinical and biochemical essential fatty acid deficiency (EFA) might occur from the feeding of eucaloric, fat-free diets was tested in two experiments in healthy men. In Study I, eight men were given fat-free, eucaloric diets containing 80% of calories as glucose and 20% as amino acid hydrolysates by a constant drip over a 24-h period. The diets were fed in succession for periods of 2 wk each, either through a superior vena cava catheter or via a nasogastric tube. EFA deficiency was detected by decreases in linoleic acid and by the appearance of 5, 8, 11-eicosatrienoic acid in lipid fractions of plasma. Linoleic acid decreased significantly during 2 wk of the fat-free diet given intravenously from 48.8 to 9.8% (percent of total fatty acids) in cholesterol esters, from 21.2 to 3.2% in phospholipids, from 9.6 to 2.0% in free fatty acids, and from 14.1 to 2.6% in triglycerides. Eicosatrienoic acid, normally undetectable, appeared 0.6% in cholesterol esters, 2.5% in phospholipids, 0.2% in free fatty acids, and 2.3% in triglycerides. EFA deficiency occurred similarly during the nasogastric feeding. In Study II a subject received the same diet continuously by the nasogastric route for 10 days followed by a 24-h fast. He was then given the fat-free diet intermittently in three meals per day for 3 days. Finally, he was repleted with a diet containing 2.6% linoleic acid. By the 3rd day of the continuous nasogastric feeding, linoleic acid had fallen significantly and eicosatrienoic acid had appeared in plasma lipid fractions as in Study I. These findings were accentuated by day 10. Adipose tissue fatty acid composition did not change. Free fatty acid outflow from adipose tissue was presumably suppressed during the 10 days of continuous feeding. With increased free fatty acid outflow during fasting and intermittent feeding, linoleic acid rose and eicosatrienoic acid decreased. After 13 days of repletion with dietary linoleic acid, the EFA deficiency readily develops when fat-free diets containing glucose are given intravenously or orally as constant 24-h infusions. These diets are similar to the hyperalimentation formulas now being used clinically.