Abstract Hepatocytes, prepared by collagenase treatment of livers of ad libitum- and meal-fed rats, were incubated with glucose, fructose, lactate, and a number of other substrates, labeled uniformly with 14C and in the presence of 3HOH. The uptake of lactate and the incorporation of 14C into CO2 and glucose equals or exceeds rates reported in perfused liver. The 14C yield in glucose from labeled fructose, glycerol, and dihydroxyacetone exceeds considerably that in CO2 and greatly that in lipids. The 14C yield in CO2 from pyruvate, lactate, alanine, and acetate is equal to or exceeds that in glucose, and the fraction incorporated into lipids is greater than that from the neutral compounds. Fatty acid synthesis is greatly increased in meal-fed as compared to ad libitum-fed animals. Incorporation of glucose and of lactate carbon into fatty acids increases with concentration up to concentrations of 100 mm. Lactate carbon is a much better fatty acid precursor than glucose carbon. When both substrates are present at physiological concentrations (10 mm in glucose and 2 mm in lactate), the incorporation of lactate exceeds several times that of glucose carbon. At concentrations below 10 mm fructose is a better precursor of fatty acids than glucose, but the reverse is true at higher concentrations. Fructose and glycerol at concentrations above 5 mm inhibit the incorporation of 3H from 3HOH into fatty acids. In cells from meal-fed rats considerable incorporation of 3H from 3HOH into fatty acids occurs in the absence of added substrate. The ratio, microatoms of 14C from substrate to microatoms of 3H from water (14C:3H ratio), in the presence of low glucose concentrations is below 0.1 and increases at 100 mm glucose up to 0.5 to 0.7. Lactate stimulates 3H incorporation up to a concentration of 20 mm, but incorporation of lactate carbon continues to increase. The 14C:3H ratio attains values close to 1.0 at about 20 mm and increases to 1.5 to 2.0 at 100 mm lactate. The incorporation of tritium from water into fatty acids by hepatocytes of meal-fed rats in the presence of 10 to 20 mm lactate ranged in most experiments from 200 to 600 µatoms per g dry weight per hour, but in a number of experiments rates above 1000 µatoms per g per hour were obtained. This corresponds to an incorporation of over 120 acetyl equivalents per g wet weight of tissue per hour. Our results indicate that free glucose has a minor role as direct precursor of fatty acids in liver, and the major substrates are glycogen and lactate. Due to the presence of glycogen and the reversibility of glycogen and glucose synthesis and glycolysis in liver, the quantitative interpretation of 14C incorporation from labeled glucose is difficult.