Coagulation Factor VIII is an acute phase protein in humans that has recently been shown to be transcriptionally responsive to interleukin-6. In this study, we have demonstrated that the human Factor VIII promoter is activated in cultured hepatocytes exposed to bacterial lipopolysaccharide (LPS). Deletion analysis has narrowed the LPS-responsive element of the Factor VIII promoter to a small region which contains two C/EBP binding sites and an adjacent NFkappaB binding site. Mutation of the downstream C/EBP site reduces LPS-responsiveness by approximately 50%, while mutation of the NFkappaB binding site completely eliminates LPS-responsiveness. While binding of C/EBPbeta and NFkappaB is still observed in gel retardation studies using acute phase nuclear extracts and a probe containing mutations to the downstream C/EBP site, neither NFkappaB nor C/EBP appear to bind to a probe in which the NFkappaB site has been mutated. Conservation of this region of the Factor VIII promoter in species which exhibit an increase in Factor VIII levels in response to inflammatory stimuli suggests that these transcription factor binding sites are important for normal regulation of the Factor VIII gene under conditions of stress.