An assay system for determination of the "fast"-acting inhibitor (antiactivator, AA) of tissue-type plasminogen activator (tPA) in human plasma was developed. The system is based on incubation of plasma samples with various amounts of vessel wall-derived tPA for 8 minutes at 25 degrees C, followed by acidification and determination of the residual tPA activity by an indirect spectrophotometric assay. One unit of AA was defined as the amount inhibiting 1 U of tPA. AA levels in normal controls (n = 26) were 1.4 to 17.4 U/ml (median 3.0 U/ml) and 0.9 to 17.5 U/ml (median 3.0 U/ml) in patients with a history of deep venous thrombosis (n = 26). When plasma was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse fibrin autography, AA activity appeared as an inhibitory band corresponding to a relative molecular mass of 50,000. In six samples the inhibitory activity of this band was directly correlated to the functional AA activity of the plasma samples.