Monoclonal antibodies (MAbs) to vascular plasminogen activator (vPA), the tissue-type plasminogen activator (tPA) in human plasma, were produced to be used as probes for immunochemical analysis. Human tissue sections and one of these MAbs were used to demonstrate the endothelial origin of plasma-tPA by immunohistochemistry. To produce MAbs, mice were immunized with semipurified vPA isolated from postocclusion human venous blood. Primed spleen cells were fused with the mouse myeloma cell line NS-1. Screening for MAb-producing hybridomas was performed with postocclusion euglobulins as a source of antigen by means of a solid-phase fibrin-vPA immunoassay. The selective and high-affinity binding of vPA for fibrin ensures the specificity and sensitivity of this test. Thus, eight hybridomas secreting MAbs to vPA were selected, cloned, and established as permanent hybridoma cell lines. Immunohistochemical analysis of cryostat sections of human tissues was performed with EA-delta 12D, a MAb having no inhibitory effect against vPA activity but binding to vPA with a high affinity. Thus, the only structures immunostained were endothelial cells of venules, capillaries, and arterioles. The EA-delta 12D monoclonal localization of plasma vPA in the endothelial lining of blood vessels provides evidence that tPA in plasma originates from the vascular wall and validates its designation as vascular plasminogen activator, ie, vPA. Also, our results are consistent with the fact that vPA in blood and tPA in tissues are immunologically identical and have a common endothelial origin.