Isolated rat liver nuclei show a substantial amount of T3 receptor release to the medium during in vitro incubation. This has been shown to be a general feature of nuclei compared after several methods of isolation and incubation. About 50% of nuclear receptors are released to the medium when incubated in sucrose-MgCl2-Tris, pH 7.85, for 2 h at 20 C.DNA, histones, and non-histone proteins (NHP) are also released. CaCl2 inhibits about 90% of the release of DNA and histones, but has less effect on inhibiting leakage of NHP and nuclear T3-binding protein (NTBP). The highest leakage for each fraction was found when incubating nuclei in the presence of EDTA. The receptor released to the medium has an affinity virtually identical to the receptor remaining in the nuclei. At least 1 mM dithiothreitol is needed to avoid degradation of the receptor. The NTBP has a sedimentation constant of 4.5 S when studied in low ionic strength gradients. Increasing KCl concentration decreases progressively its sedimentation constant, and in gradients containing 0.4 M KCl the receptor sediments as a single peak of 3.4 S. Since release of receptor to incubation medium decreases free T3 concentration, it must be taken into account in calculating receptor affinity. Total nuclear capacity in vitro is obviously underestimated, unless receptor released to medium is measured. Receptor exchange between cytosol and nucleus may be of physiologic significance.