Rat liver nuclei have recently been shown to possess limited, high affinity binding sites for triiodothyronine [T3), only a fraction of which are believed to be filled in vivo. Preincubation of nuclei in the absence of added T3 leads to a rapid decrease in binding capacity. This loss of binding sites is considerably faster at 37.degree. C than at 4.degree. C. Once binding equilibrium is reached between nuclear bound and free T3 the level of binding does not change during the time in which a considerable loss of binding had occurred when nuclei were preincubated in the absence of hormone. The rate of dissociation of T3 from nuclei is slower than the rate of loss of binding sites during preincubation in the absence of added hormone. It appears as if unfilled sites are being lost during the preincubation period. Administration of cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, respectively, to rats caused a decrease in the amount of T3 which could be bound in the in vitro binding assay to liver nuclei.