A study of the human clotting factor IX promoter by DNase I footprinting and gel shifts in vitro, and by functional analysis of HepG2 cells in vivo, suggests that the liver-enriched transcription factor, HNF4, is involved in transactivating two cis-acting elements, i.e. X (nucleotides -15 to +3) and Y (nucleotides +15 to +36), in addition to the well-known element centred around nucleotide -20. Other members of the orphan receptor superfamily, e.g. ARP1 and COUP/Ear3, repress the factor IX promoter possibly by competition with HNF4 binding sites in the X and Y elements, but probably not at the -20 element. Mutations at -6 in the promoter, similar to those found in patients with haemophilia B, hinder HNF4 binding and transactivation of the X element, suggesting that impaired HNF4 binding contributes to the down-regulation of the factor IX expression in these patients, but is unlikely to be the only factor involved.