Protein S is expressed in a number of tissue types, one of the most physiologically relevant being the liver. However, transcriptional control of protein S gene expression is poorly understood. We have characterised a 638 bp area in the 5' flanking region of the human protein S gene, spanning all 10 previously reported transcription initiation sites, which demonstrates promoter activity in the human liver-derived cell line HepG2. More refined reporter gene analysis of this region enabled the identification of three transcription initiation sites whose absence is associated with significantly reduced promoter activity, together with a number of positively and negatively acting transcriptional regulatory elements. Consistent with these findings, DNaseI footprinting analysis identified eleven sites (I-XI) from within this 638 bp region that show evidence of binding nuclear proteins. We present evidence to show that the liver-specific factors hepatocyte nuclear factor 1 (HNF1) and HNF4 bind regions of the protein S promoter, which lie within the identified protein binding sites V and VIII, respectively, and that HNF4 activates the protein S promoter. Reporter gene analysis suggests that members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors are potent activators of protein S gene transcription in HepG2 cells.