The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic ␣-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128 -366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360 -366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo-and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.The nuclear receptor superfamily comprises a large set of ligand-regulated transcription factors. This superfamily includes receptors for steroid hormones, retinoids, thyroid hormone, and vitamin D 3 , as well as a large number of structurally and functionally related transcription regulatory proteins whose natural ligands are not yet known, the so-called orphan receptors (reviewed in Refs. 1 and 2). Nuclear receptors exhibit a modular structure with six distinct regions (referred to as regions A-F), which correspond to functional domains. The N-terminal region A/B is highly variable among nuclear receptors and contains a ligand-independent transactivation function AF-1 (2). Region C contains a highly conserved DNA binding domain (DBD) 1 composed of two zinc-coordinated modules and is responsible for specific binding to cognate response elements (Refs. 1 and 2 and references therein). The exact functions of regions D and F are not clear, although they appear to be well conserved for each receptor across species. Region D is postulated to function as a flexible hinge between the DBD and the ligand-binding domain (LBD), allowing rotational differences between these domains when dimeric receptors bind to direct, inverted, or palindromic repeats (2). Interestingly, the D regions of the thyroid hormone (TR) and retinoic acid receptors (RARs) interact with ...