A simple and discriminating assay for the determination of the fast-acting plasminogen activator inhibitor (PAI) activity in human plasma is described. The method is based on the inhibition of purified tissue plasminogen activator (tPA) by plasma diluted with a PAI-depleted plasma and the subsequent measurement of residual tPA in the presence of CNBr-fibrinogen fragments, purified plasminogen and a plasmin sensitive chromogenic substrate CBS 10.65. This assay does not require any acidification step, and allows PAI determination directly on plasma. Since dilutions are made in PAI-depleted plasma, all the serine-protease inhibitors, except PAI, are kept constant in their effect on the assay. Thus, any detectable degree of inhibition can only be ascribed to PAI. Under these conditions, parallel titration curves of tPA are obtained in plasma and the values of PAI are reproducible when measured at different dilutions. The PAI levels of 31 normal volunteers ranged from 0.3 to 8.7 IU/ml (mean: 3.5 IU/ml). After venous occlusion, variations of PAI were associated with the release of tPA. A marked increase of PAI levels was observed in the post-operative period and in pregnancy. In this case both PAI-1 and PAI-2 related activities were measured. Due to its simplicity, the assay can be easily used for the screening of patients with thrombotic diseases.