[3H]Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and nucleus. We studied the [3H]-TRH binding capacity of a highly purified nuclear fraction isolated by an original procedure from GH3 cells. The nuclei still presented their double nuclear envelope. They are able to bind [H]-TRH to the same extent as nuclei isolated from GH3 cells previously exposed to [3H]-TRH. The equilibrium of binding was reached after 2--5 min incubation at 25 degrees or 35 degrees C. The binding is stable at 4 degrees C and partially (50%) dissociated within 15 min at 25 degrees C. 50% of the binding was inhibited by large excess of unlabelled TRH. Nuclei obtained from a variant GH3 cell which has lost its responsiveness to TRH presented only the noncompetitive binding compartment. The binding was found dose dependent and not saturable. Two apparent dissociation constants were evaluated: 1.5--2.5 x 10--8M and 2.10--6M, respectively, for high and low doses of [3H]-TRH. The first one was identical to that previously found for intact GH3 cells. The present data show the existence of specific nuclear binding sites for TRH, establish their characteristics and suggest a possible nuclear site of action for that peptide hormone.