The binding of thyroxine (125I-T4) in rat liver homogenate and isolated nuclei was investigated in vitro. In homogenate, the binding of T4 was modified by thiocyanate ion (SCN-) added to the incubation mixture as potassium thiocyanate. The displacing effect of SCN- increased with increasing concentrations of this compound, the lowest effective dose being that which significantly displaced T4 from liver homogenate (105 pmol per one ml medium). Moreover, SCN- significantly increased the displacing effect of unlabelled T4. In contrast, however, no effect of SCN- was found on the binding of T4 in the isolated liver nuclei. In sonicated nuclei no effect of SCN- on T4 binding was found, but specific binding of T4 under these circumstances was impaired. Finally, in a homogenate a displacing effect of other univalent anions (BF4-, ClO4- and MnO4-) was found resembling that of SCN-. It is suggested that, in the liver homogenate, SCN- displaces T4 from non-specific binding sites. This view is also supported by the fact that in liver nuclei, where specific binding sites prevail, no effect of SCN- was observed.