The nuclear binding of the activated glucocorticoid-receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI-I, MTI-II and MTI-III). In this study, we purified the most potent of the three species (MTI-II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two-dimensional PAGE. Purified MTI-II inhibits GR binding to DNA containing glucocorticoid-response elements. To obtain the amino acid sequence of MTI-II, we digested the MTI-II with endopeptidases. The N-terminal amino acid sequences of the four digested fragments indicated that MTI-II is an 11.5-kDa Zn 2+ -binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI-II (ZnBP, parathymosin) and that GR binds to the MTI-II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.Keywords: glucocorticoid receptor; nuclear-translocation inhibitor; parathymosin; purification; zinc-binding protein.When nuclei or DNA were incubated with increasing concentrations of cytosol containing activated (transformed) glucocorticoid receptor (GR), receptor binding to nuclei or DNA reached apparent saturation [1±4]. Several laboratories have reported that this pseudo-saturation is due to the presence of an endogenous macromolecular inhibitor (or inhibitors) in the cytosol of rat liver [1±5] and rat hepatoma cells [6]. With regard to the physiological role of these inhibitors, it is speculated that the target cells may have an inherent mechanism that regulates the interaction of GR with the genomic machinery and thereby controls hormone responses at the cellular level [2,5]. Alternatively, the inhibitor may play a role in the release of the receptor from its nuclear-binding state [5]. We found that AH 130 ascites tumor cells (glucocorticoidresistant cells) have approximately five times more inhibitory activity than cells from rat liver [7], and that the inhibitory activity in regenerating rat liver following 70% hepatectomy is increased significantly during periods of DNA synthesis and subsequent mitosis and then decreases to normal level [8]. Thus, the inhibitors may be involved in cell proliferation by inhibiting GR binding to nuclei.Previously, we separated the macromolecular-translocation inhibitors (MTIs), which inhibit GR binding to nuclei in vitro, from the cytosol of rat liver [9] and other cells [7,8] into at least three components (MTI-I, MTI-II and MTI-III). Recently, we purified MTI-III to apparent homogeneity and characterized i...