An enzyme-linked immunosorbent assay for free tissue-type plasminogen activator (t-PA) in human blood was developed based on a murine monoclonal antibody directed against the active site of t-PA. The lower limit of sensitivity of the assay applied to plasma is 2 ng/mL for one-chain t-PA but only 100 ng/mL for two-chain t-PA. Free t-PA in plasma taken at rest was found in 6 of 21 healthy subjects (4.5 +/- 0.8 ng/mL, mean +/- SD) and increased to 14 +/- 7.0 ng/mL after venous occlusion in 18 of these individuals. A linear correlation between total t-PA and free t-PA was observed with r = 0.92 (n = 18) and a slope of 1.08, indicating that t-PA released from the vessel wall circulates in the blood as the one-chain form. In 16 of 18 patients with deep vein thrombosis, the increase of total t-PA antigen after venous occlusion was comparable to that observed in controls, but the free t-PA was significantly lower or undetectable. The present assay for free t-PA may be useful for the investigation of the release and inhibition of t-PA under physiological, pharmacological, or pathological conditions in humans.