Assay methods for the detection of both tissue factor pathway inhibitor (TFPI) function (two-stage chromogenic assay) and for TFPI antigen levels (competitive ELISA) have been developed and applied to the measurement of TFPI in normal plasma, in post-heparin plasma and to recombinant TFPI. There was good correlation in TFPI levels, measured using the two methods (r = 0.848; P < 0.001) in the normal plasma samples (n = 21) with the values ranging from 0.6 to 1.4 units per ml relative to a normal reference plasma pool (assigned 1.0 unit per ml). The post-heparin plasma samples were associated with increased levels of both TFPI functional activity and antigen. However, there was poor correlation between the two methods, with an increase in antigen levels greatly exceeding the increase in functional activity. This discrepancy was also found with recombinant TFPI and may reflect the different responses of the two assay methods to lipoprotein-bound TFPI (in the normal plasma reference) and the 'free' TFPI in post-heparin plasma and recombinant TFPI. These findings have implications in the choice of suitable reference materials for the assay of TFPI.