A study of the effect of thyroxine in vitro on fatty acid synthesis in rat liver supernatant and microsomal preparations has been undertaken. High concentrations of thyroxine inhibit the supernatant de novo synthesis of the major reaction products, myristic and palmitic acids. Similar inhibition using either [14C]—acetyl—CoA or [14C]—malonyl—CoA as substrate indicates that inhibition is beyond the acetyl—CoA carboxylase reaction. The influence of lower concentrations of thyroxine on the aerobic and anaerobic incorporation of [14C]—malonyl—CoA into microsomal lipids was studied separating the reaction products by gas—liquid and thin—layer chromatography. These studies indicate that thyroxine stimulates the microsomal desaturation reactions but has no effect on the chain elongation reactions. The stimulation of microsomal desaturation may be at least partially due to the antioxidant effect of thyroxine. These described in vitro effects of thyroxine are similar to previously described in vivo studies which demonstrate that thyroxine promotes unsaturated fatty acid accumulation because of its stimulatory effect on the desaturation reaction (Gompertz, D., and A. Greenbaum, Biochim Biophys AdaL. 116: 441, 1966). (Endocrinology91: 1481, 1972)