1. Fatty acids have the capacity for inhibition of nuclear T3 binding (INB). The present studies were undertaken to describe the INB-activity of fatty acids as a function of chain length, unsaturated bonds, cis-trans configuration, and esterification. 2. Isolated rat liver nuclei were incubated with [125I]T3 in the absence or presence of fatty acids in concentrations of 0.011, 0.033, 0.1 and 0.3 mM respectively. 3. INB-activity depended on the chain length, being greatest at 14 carbon atoms. 4. INB by unsaturated fatty acids was greater than that of saturated fatty acids, and increased with increasing number of double bonds. 5. Fatty acids in the cis configuration had greater INB-activity than those in trans configuration. 6. Esterification of fatty acids decreased INB-activity: monoglycerides still had some effect, but di- and triglycerides had no effect.