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The pathway of anaerobic glycolysis of Trypanosoma brucei has been studied by the following five approaches : 1. Ensyme activity required for the various different postulated schemes for anaerobic glycolysis (hexose monophosphate aldolase, glycerol dehydrogenase, glycerophosphate : glucose or glycerophosphate : fructosesphosphate or glycerophosphate : triose or* glycerophosphate : ALP transphosphorylases) have been examined in a variety of assay conditions. Only significant activities of glycerophosphate : ALP transphosphorylase were detectable. 2. Broken cell incubation studies established that there were no significant differences in the sequence of the increase and decrease in glycolytic intermediates between aerobic and anaerobic pathways.3. Whole cell adenylate charge and glycolytic intermediates were assayed in steady-state aerobic, anaerobic and in the transitions between aerobic to anaerobic, and anaerobic to aerobic showed glucose-6-phosj)hate production to be the rate limiting step in anaerobic glucose utilization. Salicylhyaroxamic acid (0.5 mM) inhibited only glycerophosphate oxidase -and so simulated anaerobiosis. 4. The inhibitory effect of glycerol on whole cells metabolising glucose anaerobically showed it to be dependent upon the intracellular concentration of glycerophosphate. Consequently its inhibitory effect is not caused through the inhibition of glucose transport. 5. The concentration of glycerophosphate in cells metabolising glucose under glycerophosphate oxidase inhibited conditions was found to increase rapidly to a concentration that was independent of time and extracellular glycerol concentration. Furthermore it was found to be an intermediate in anaerobic glucose utilization. The results of this work were consistent with the pathway of carbohydrate metabolism under glycerophosphate oxidase inhibition which involved the production of glycerol plus ATP from ADP plus glycerophosphate catalysed by glycerokinase.