The possibilities that the hypotriglyceridemic effect of clofibrate involves activation of carnitine-dependent oxidation of fatty acids in liver and that this may be partially mediated through thyroxine have been examined. 0.25% clofibrate in diet for 10-15 days, was found to increase carnitine 3-fold in livers of male as well as female rats. Liver carnitine was nearly doubled by L-thyroxine, 6 mg/kg of diet fed for 10 days, and so was the activity of gamma-butyrobetaine hydroxylase. Clofibrate decreased carnitine in heart and urine; thyroxine did not affect these parameters but increased serum carnitine by 26%. Clofibrate feeding doubled the concentration of hepatic long-chain acyl(-)carnitine, mitochondrial carnitine, and the rate of mitochondrial carnitine-acylcarnitine translocase reaction, and enhanced acetoacetate production in liver homogenates as well as mitochondrial oxidation of palmitoylcarnitine in the presence of malonate. The ratio of esterified to free carnitine in urine and serum was also increased by clofibrate. These results suggest that clofibrate and thyroxine may exert their hypotriglyceridemic effect, in part, through the activation of carnitine-mediated transport of fatty acids in liver mitochondria.