A sandwich enzyme-linked immunosorbent assay (ELISA) for determination of tissue-type plasminogen activator (t-PA) was developed. 96-well flat-bottom polystyrene plates were coated with polyclonal (goat) anti t-PA IgG (10 micrograms/ml). After addition of samples monoclonal (murine) anti t-PA IgG (1.5 micrograms/ml) was added. Finally, peroxidase labelled anti-mouse IgG (goat) was used to quantify the bound second antibody. The assay can be used for determination of t-PA antigen in purified systems, in cell culture supernatants, and in human plasma, provided that EDTA (0.005 M) is present in the sample. In 78 healthy volunteers, t-PA antigen levels at rest were 0.4 - 15.2 ng/ml (4.3 +/- 2.7, means +/- S.D.); A significant positive correlation between t-PA antigen and age could be demonstrated.