Long-chain fatty acids are oxidized by intact rat kidney mitochondria under experimental conditions which permit the location of three long-chain acyl-CoA synthesizing systems.Oleate oxidation which is 2,4-dinitrophenol-insensitive and which is inhibited by arsenate and excess phosphate, is probably energized by GTP. The isolation of the GTP-dependent acylCoA synthetase from sonicated kidney mitochondria supports this hypothesis.Oleate oxidation which is insensitive to phosphate and arsenate and requires oligomycin, utilizes "endogenous" ATP and is not linked to added carnitine.The third oleate oxidation system requires added ATP, CoA and phosphate and depends on a carnitine-linked transport mechanism.The prcsence of three acyl-CoA synthesizing systems have been postulated in intact rat liver mitochondria [l-71. The first one depends on substrate level phosphorylation and is inhibited by phosphate, arsenite and arsenate [1,2]. This system has been shown to be identical [I, 21 to the GTP-dependent acyl-CoA synthetase described both in beef liver IS] and in rat liver mitochondria [9]. The second system would derive the energy from "endogenous" ATP [1][2][3][4]6,7] and is thought to be "within" the mitochondrion. Acyl-CoA synthesized by this system does not depend on a carnitine-linked transport mechanism [lo] for its oxidation. The third acyl-CoA synthetase system would derive its energy from "exogenous') ATP C5-71 and is thought to occur "externally" or a t the surface of the mitochondrion. In this case, acyl-CoA must be converted to the carnitine ester for acyl transport to the "internal" site of p-oxidation.Van den Bergh [ill suggested that the acyl-Con synthesizing system which derives its energy from "endogenous" ATP is present in liver mitochondria, whereas in both kidney and heart mitochondria free long-chain fatty acids are oxidized only in the presence of added carnitine. Bode and Klingenberg [12] proposed that the activation of long-chain fatty acids in muscle mitochondria involves the transfer of carnitine from acetyl -carnitine to acyl-residues.Furthermore, no GTP-linked activation would be present in kidney 1113, heart 1111 and brain mitochondria [12].I n the present communication we report experiments which are relevant to the problem of longchain fatty acid oxidation in rat kidney mitochondria. Oxygen uptake was measured with a Clark oxygen electrode as described by Kielley and Bronk LlS].Acyl-CoA synthetase and succinic acid thiokinase were routinely assayed by measuring the rate of CoA sulfhydryl group disappearance [16]. The GTPdependent acyl-CoA synthetase and the succinic acid thiokinase were tested in a system containing 0.4 M Tris-HC1, pH 7.4, 0.3 M sucrose, 5 mM KBH,, 5 mM MgCl,, 2.5 mM GTP, 1 mM CoA and either 25 mM potassium octanoate, 2.5mM sodium oleate or 125mM sodium succinate. The total volume was 0.2 ml. The activity of the ATP-dependent acyl-CoA synthetase was measured in the presence of 5 mM ATP under similar conditions except that the Tris buffer was 50 mM and sucrose was repl...