A relationship between non-esterified fatty acids (NEFA) in plasma and the metabolism of glucose was investigated. NEFA levels in plasma increased after heparin injection, likely due to lipase activity. However, after a mixed meal, NEFA levels fell sharply and remained below fasting levels for 2 or more hours. Fat alone did not significantly affect NEFA levels, while glucose and insulin caused a decrease similar to that after a mixed meal, and epinephrine caused a marked increase. A new method for measuring NEFA was developed, which is rapid, simple, and has a low coefficient of variation. It is specific for NEFA but not absolutely reliable. The method involves extracting lipids into a one-phase system and converting it to two phases. The extraction mixture consists of isopropyl alcohol, heptane, and sulfuric acid, while the titration mixture includes thymol blue and ethanol. The method was tested for reproducibility and showed good results. The results showed that NEFA levels varied with fasting, mixed meals, glucose, insulin, and epinephrine. Fasting increased NEFA levels, while glucose and insulin decreased them. Epinephrine increased NEFA levels. Corn oil did not significantly affect NEFA levels even when enough was consumed to produce lipemia. NEFA levels were normal in some patients with elevated plasma lipids. The study supports the belief that most of the acidity determined by the method resides in albumin-bound fatty acids, with little hydrolysis of triglycerides occurring during extraction or titration. The method is more rapid and sensitive than previous techniques. The work was aided by Dr. E. H. Ahrens, Jr. and Dr. Rodes Trautman.A relationship between non-esterified fatty acids (NEFA) in plasma and the metabolism of glucose was investigated. NEFA levels in plasma increased after heparin injection, likely due to lipase activity. However, after a mixed meal, NEFA levels fell sharply and remained below fasting levels for 2 or more hours. Fat alone did not significantly affect NEFA levels, while glucose and insulin caused a decrease similar to that after a mixed meal, and epinephrine caused a marked increase. A new method for measuring NEFA was developed, which is rapid, simple, and has a low coefficient of variation. It is specific for NEFA but not absolutely reliable. The method involves extracting lipids into a one-phase system and converting it to two phases. The extraction mixture consists of isopropyl alcohol, heptane, and sulfuric acid, while the titration mixture includes thymol blue and ethanol. The method was tested for reproducibility and showed good results. The results showed that NEFA levels varied with fasting, mixed meals, glucose, insulin, and epinephrine. Fasting increased NEFA levels, while glucose and insulin decreased them. Epinephrine increased NEFA levels. Corn oil did not significantly affect NEFA levels even when enough was consumed to produce lipemia. NEFA levels were normal in some patients with elevated plasma lipids. The study supports the belief that most of the acidity determined by the method resides in albumin-bound fatty acids, with little hydrolysis of triglycerides occurring during extraction or titration. The method is more rapid and sensitive than previous techniques. The work was aided by Dr. E. H. Ahrens, Jr. and Dr. Rodes Trautman.