An enzyme-linked immunosorbent assay (ELISA) was developed for the specific detection of human angiostatin-like plasminogen moieties (comprising kringles 1-4) in biological samples. The assay involves prior removal of all other plasminogen moieties by immunoadsorption of diluted samples (to about 10 ng/ml plasminogen) with a mixture of insolubilized MA-42B12 (directed against kringle 5) and MA-31E9 (directed against the proteinase domain). The recovery of angiostatin during this procedure is > or = 95%. Subsequently, angiostatin-like fragments are detected in an ELISA, based on two monoclonal antibodies reacting with nonoverlapping epitopes in the kringle 1-3 domain: MA-36E6 for capture and MA-34D3 for tagging. The assay has a lower detection limit of about 0.1 ng/ml and is performed with intra- and interassay coefficient of variation of 2.4% and 15%. In tumor fluids obtained from cancer patients (n = 10), angiostatin levels ranged between 0.24 and 6.7 microg/ml (1.62+/-0.60 microg/ml; mean+/-S.E.M.) The identity of angiostatin was confirmed by immunoblotting using specific monoclonal antibodies. A weak correlation (r = .66) was observed with the total plasminogen concentration in these samples. This ELISA thus appears suitable for the specific quantitation of angiostatin-like plasminogen moieties in biological samples, and may be useful to study its (patho)physiological relevance.