An improved assay method for tissue plasminogen activator (t-PA) in plasma samples is described, which allows the plasma t-PA activity to be determined within 3 hours. The method minimizes the action of possible inhibitors by immediate acidification of blood samples prior to separation of the blood cells. In almost all plasma samples variable amounts of a rapid t-PA inhibitor could be demonstrated. The inhibitor was present in plasma and seemed not to be associated with the blood cells. A quantitative method for determination of the inhibitor content in plasma was developed. In this investigation 1 unit of inhibitor is defined as the amount that neutralized 1 unit (0.06 pmol) of t-PA in 10 minutes. The content of t-PA inhibitor in plasma samples from healthy individuals was determined as 0.6 U/ml +/- 0.5 U/ml (range 0-3.2, median 0.4). In a group of patients (n = 75) with suspected disturbance of the haemostatic system, elevated levels were found (3.8 +/- 2.7, U/ml median 3.6 U/ml, range 0 - 15 U/ml). Kinetic analysis with plasma from three patients with elevated inhibitor content showed very similar rate of t-PA inhibition and assuming the formation of a 1:1 t-PA-inhibitor complex a second order rate constant of 10(7) M(-1) s(-1) was calculated. A regulatory function may be anticipated for this inhibitor, but its exact patophysiological role has still to be established.