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1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.