When washed spleen slices from fed rats are incubated with 3 mm-[U-14C]glucose, the rate of glucose utilization (46.2 mumol/h per g dry wt.) is sufficient to account, theoretically, for 80% of the O2 consumption. Measurement of net lactate production, however, and the fate of the radioactive carbon, indicates that the contribution of glucose to the respiratory fuel of the tissue is only 25-30% whereas 60-70% of the glucose utilized is converted into lactate. At saturating glucose concentrations (above 5 mm) its contribution to the respiratory fuel of the slice is increased to a maximum value of 34-39%. Only 2% of the glucose utilized is metabolized via the oxidative steps of the pentose phosphate pathway. Starvation for 72 h marginally increases both the rate of glucose utilization (by 21%) and its net contribution to the respiratory fuel (by 29%). Insulin, glucagon, adrenaline and adenosine 3':5'-cyclic monophosphate have no significant effect on either the rate of glucose utilization or on the pattern of radioactive isotope distribution. The uptake of glucose is increased by only 20%, whereas the production of lactate doubles when slices are incubated under anaerobic conditions. In assessing the suitability of spleen slices for metabolic studies, the only serious major perturbation, compared with the freeze-clamped organ, is an elevated mitochondrial [NAD+]/[NADH] ratio (connected with increased endogenous NH3 production) that is partially restored to normal values on incubation with glucose. Equal proportions of erythrocytes and leucocytes are found in the washed spleen slice. Metabolic contributions of the constituent cell populations in the washed slice are calculated and it is concluded that lymphocytes account for the major part of the glycolytic metabolism (80-90%), whereas the contribution of erythrocytes is insignificant.