Hepatic nuclear factor 4 (HNF4) is a transcription factor whose expression is crucial for mouse embryonic development, for liver-specific gene expression and for the prevention of one form of maturity-onset diabetes of the young. Its domain structure has been defined previously and is similar to other members of the nuclear receptor superfamily. A repressor domain has now been localised to a region of 14 amino acids (residues 428-441) near the C-terminus of HNF4 and is sufficient by itself to repress the activity of the activation function 2 (AF2) domain. Multiple mutations within this repressor domain enhance activity. Interestingly, this repressor domain shares homology with a repressor domain in the progesterone receptor. In a detailed mutagenesis study of the AF2 core, we demonstrate that L 366, which is conserved in the AF2 core between HNF4 and a number of orphan nuclear receptors, is essential for the full activity of the AF2 domain. Furthermore, a double mutation of E 363 and L 366 suggests that these residues might act in a cooperative manner.