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International audience; In its blood stages the malaria parasite, Plasmodium, displays very high lipid metabolism. We present evidence for an abundant long-chain acyl-CoA synthetase (EC 6.2.1.3) activity in Plasmodium knowlesi-infected simian erythrocytes. The activity was found to be 20-fold higher in the schizont-infected (the last parasite stage) than in control erythrocytes. The cosubstrate requirements of the enzyme were similar to those previously reported for acyl-CoA synthetases from other sources. Among the separated reaction products of oleyl-CoA synthetase, only PPi and oleyl-CoA were inhibitory, with Ki over 350 μM. The fatty acid specificity of the parasite acyl-CoA synthetase activity was fairly marked and depended on the unsaturation state of the substrate. The tested fatty acids displayed similar Vmax, whereas their Km ranged from 11 (palmitate) to 59 μM (arachidonate). Finally, experiments involving heat inactivation and separation on hydroxyapatite excluded the presence of a specific arachidonyl-CoA synthetase identical to those present in other cells. On the other hand, fatty acid competition experiments evidenced the existence of at least two distinct enzymatic sites for fatty acid activation in P. knowlesi-intected simian erythrocytes: one is specific for saturated fatty acids and the other for polyunsaturated species, whereas oleate could be activated at both sites.