The presence of activators of the fibrinolytic system in blood plasma has been assumed for a long time but never convincingly documented by the isolation of characterized and physiologically plausible enzymes. The low catalytic efficiency of previously identified plasma plasminogen activators, which has made their physiological significance uncertain, prompted us to search for other plasma enzymes, resembling especially the potent urinary activator, urokinase. We report here the detection of a urokinase-like activity in human plasma, and the isolation of the enzyme from whole plasma protein fractions. The purified enzyme is indistinguishable from the 53,000-dalton components of human urinary urokinase in the following respects: apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double immunodiffusion, amino acid analysis, two-dimensional tryptic peptide maps, catalytic efficiency with synthetic peptide substrates, and inhibitor spectrum. The results suggest (a) that the enzyme is present in plasma in a latent form whose nature remains to be defined, and (b) that the circulating concentration is at least 5 to 10 microgram/liter, and sufficient to generate substantial levels of plasmin, particularly if activation were somehow confined by localization at specific sites.