The effect of in vitro addition of three hypolipidemic drugs (clofibric acid, bezafibrate and gemfibrozil) on rat palmitoyl-CoA hydrolases has been studied, by using a spectrophotometric method (Berge RK, Biochim Biophys Acta 574: 321-333, 1979) optimized for valoration of crude enzyme preparations. Mitochondrial and microsomal hepatic palmitoyl-CoA hydrolase activities were inhibited by the three drugs in a concentration-dependent fashion. The order of inhibitory potency was gemfibrozil greater than bezafibrate greater than clofibric acid, irrespective of the enzyme activity tested. Cytosolic rat brain palmitoyl-CoA hydrolase activity was not affected. Kinetic studies with gemfibrozil on the solubilized microsomal palmitoyl-CoA hydrolase activity point to a mixed non-competitive type of inhibition.