T he site of synthesis of Hageman factor (HF, Factor XII) has not been previously demonstrated with certainty. We have studied the production and release of HF in the isolated perfused rat liver and have compared rates of synthesis in this system with absolute rates of degradation measured in vivo. Rat livers, perfused for 5 h with a recycling fluid consisting of a perfluorochemical emulsion (Fluosol 43), were used to demonstrate a cumulative increase of HF in the perfusate as measured by a specific and sensitive radioimmunoassay. The rate of increase in the perfusate pool of HF during the final 4 h of perfusion yielded a mean synthetic rate of 3.5 micrograms/h per 100 g body wt, which was approximately 0.2% of the synthetic rate of albumin in the same system. The cumulative appearance of albumin and transferrin was linear after 1 h and calculated rates of synthesis were 2,012 micrograms/h per 100 g and 263 micrograms/h per 100 g body wt, respectively. De novo synthesis of HF was confirmed by demonstrating incorporation of [14C]lysine into specific immunoprecipitates of HF, and by the observations that both specific incorporation of labeled amino acid and net release of immunoassayable HF were inhibited by the administration of cycloheximide. Finally, it was evident that the rates of synthesis observed in the isolated perfused liver agreed closely with absolute rates of degradation of HF measured in vivo with 125I-rat HF (4.0 micrograms/h per 100 g). From these data we conclude that the liver is the principal site of synthesis of HF.