A functional, immunoradiometric assay for a specific plasminogen activator inhibitor (PAI) was developed. This assay was based on the ability of the PAI to bind rapidly and strongly to immobilized tissue-type plasminogen activator (tPA). The extent of binding was quantified by incubating the PAI-tPA complex first with rabbit antiserum to the PAI and then with 125I-labeled goat anti-rabbit IgG. The interaction between tPA and the PAI was rapid, time- and concentration-dependent, sensitive over a broad range of PAI concentrations (1 to 100 ng/ml), and competed by urokinase but not streptokinase. The widespread application of this new technique was indicated by its ability to detect an immunologically related PAI not only in a number of other cell types, but also in platelets from which it can be released by thrombin, and in blood. This assay thus provides a quantitative approach for assessing the role of this PAI in a variety of fibrinolytic processes.