1. The metabolism of glucose labeled uniformly with 14C, and in positions 2, 3 and 5 with tritium by hepatocytes from fed and fasted rats were studied. Cells were incubated with glucose as sole substrate, or with glucose and a variety of glucose precursors, and uptake or production of glucose, and the utilization of the isotopes was determined. 2. There was no uptake of glucose at concentration of up to 15 mM, and net glucose synthesis in the presence of precursors. 14C was however recovered in CO2, lactate and amino acids, and tritium in water. Considerable incorporation into glycogen from 14C and 3H-labeled glucose occurred at high (above 20 mM) glucose concentrations. 3. The yield in water always exceeded that in 14C-labeled products. The yield in 3HOH from [2-3H] glucose exceeded that from [5-3H] glucose, and the latter was greater than from [3-3H] glucose. 4. Utilization of labeled glucose does not follow Michaelis-Menten kinetics. The fractional rate of uptake of 14C and tritium-labeled glucose increases with glucose concentration with a maximum at about 15 mM and then declines. 5. The effect of numerous gluconeogenic substrates on the isotope utilization and the 3H/14C ratio in glycogen was studied. The uptake of 14C was always depressed. Addition of lactate and dihydroxyacetone has little effect on the detritiation of [2-3H] glucose, but it is depressed by other substrates. The detritiation of [3-3H]-and[5-3H]glucose is depressed in gluconeogenesis, that from [3-3H]glucose usually more than from [5-3H]glucose. In the presence of lactate detritiation of [3-3H]glucose is about half that from [5-3H]glucose. 6. Equations to calculate the phosphorylation of glucose and fructose 6-phosphate in the presence of futile cycling between glucose and glucose 6-phosphate and fructose 6-phosphate and fructose 1,6-bisphosphate were derived. 7. The estimate of glucose phosphorylation requires determination of the specific activity of glucose 6-phosphate from [2-3H]glucose. It appears that futile cycling between glucose and glucose 6-phosphate is extensive in cells with a high glycogen content, but is low in cells from starved rats and nearly absent in those from diabetic animals. 8. The estimation of the phosphorylation of fructose 6-phosphate in the presence of cycling requires knowledge of the specific activities of fructose 6-phosphate and fructose 1,6-bisphosphate from [3-3H]glucose. At present there are no adqquate data to calculate phosphorylation and recycling of fructose 6-phosphate, but under some conditions the rate may be quite high.