1. Gluconeogenesis was studied in isolated avian hepatocytes. The highest rate of glucose production obtained was from lactate, followed by dihydroxyacetone, glyceraldehyde, and fructose. Alanine was converted to glucose at only about 4% the rate of lactate. 2. Addition of 10 mM sorbitol, xylitol, or ethanol to the hepatocytes increased glucose production from pyruvate 25-40%, while glycerol addition increased it only 9%. 3. Addition of beta-hydroxybutyrate had no effect on glucose production from lactate or pyruvate. 4. Addition of octanoate had no effect on glucose production from pyruvate, but depressed it from lactate at 5 mM. 5. Differences in the formation of glucose from various substrates suggest some basic differences in the mode of glucose production between the chick and the rat and guinea-pig.