The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma- derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.