The subcellular distribution of the acyl CoA synthetases of rat liver was reinvestigated in order to determine whether part of this activity occurs in peroxisomes. Rat liver was fractionated by differential centrifugation and by equilibrium density centrifugation. Acyl-CoA synthetase was assayed using a new, simple extraction procedure on 3 substrates: palmitate, laurate and octanoate. Comparison of the resulting synthetase distributions with the distributions of marker enzymes for peroxisomes, mitochondria and endoplasmic reticulum demonstrated the presence of some synthetase activity in each of the 3 organelles. These trimodal synthetase distributions were evaluated quantitatively by means of a computer program that calculated optimal linear combinations of marker enzymes using a least squares criterion. Peroxisomes contained 7% of the liver''s palmitoyl-CoA synthetase activity and 6% of its lauroyl-CoA synthetase activity, but no demonstrable octanoyl-CoA synthetase activity. The remainder of these activities are divided between the mitochondria and endoplasmic reticulum, in agreement with previous studies. The chain length specificity of the synthetase(s) of each organelle appears to be unique. The absolute activity of the peroxisomal palmitoyl-CoA synthetase is sufficient to maintain maximal peroxisomal .beta.-oxidation. Clofibrate treatment of the rats caused a 2.6- to 3.1-fold increase in the liver''s total acyl-CoA synthetase activities. The subcellular distribution was not greatly affected by this drug treatment.