This article describes a two-step bio-immuno assay (BIA), which determines active (free) tissue-type plasminogen activator (t-PA) in (acidified) plasma. Plasma samples, diluted in buffer of pH 6.0, are added to the wells of a microtiter plate containing an immobilised monoclonal antibody which binds t-PA without affecting t-PA activity. After an overnight incubation at 4°C, bound t-PA activity is determined by incubating the wells at 37°C with plasminogen, CNBr-digested fibrinogen and D-Val-Leu-Lys-pNA, followed by measurement(s) of the absorbance (A) at 405 nm at timed intervals. The ΔA/t2 or, alternatively, the AA after 4 hours (endpoint determination) give an accurate value for the active t-PA concentration. The assay has a lower detection limit of 3 pg t-PA/ml and does not discriminate between various forms of active t-PA. The intra-assay coefficients at 0.2 and 0.05 ng t-PA/ml were 5.5% and 4.3%, respectively. Active t-PA values in samples (N = 66) determined with this assay and with the t-PA BIA from Chromogenix correlated relatively well (r = 0.78). Within a subset of these samples (N = 18), a negative correlation with both t-PA/PAI-1 (r = 0.79) and PAI-1 antigen (r = 0.78) values was observed. Surprisingly, no correlation was found with values for t-PA antigen (r = 0.21).