T hree kinds of plasmin were found to be generated when plasminogen or [¹²⁵I]plasminogen was incubated at 32°C for longer than 20 min in urokinase and 50% glycerol. Each plasmin was then separated by G-200 or G-75 Sephadex filtration, and its physicochemical properties were determined. The molecular weights of the three plasmins as determined by G-200 Sephadex filtration were 125,000±5,000(SD), 63,000±2,000(SD), and 31,500±1,000(SD), and those by sodium dodecyl sulfate(SDS)-polyacrylamide electrophoresis were 130,000±5,000(SD), 64,000±3,000(SD), and 32,000±1,500(SD). It was also found that during the incubation of the smallest plasmin in SDS and beta-mercaptoethanol it was further split into two smaller pieces of about 16,000 mol wt and that polymer proteins of 95,000±2,000(SD) and 48,000±1,500(SD) mol wt were formed. Despite these differences in the molecular size of the three plasmins, the specific activity of each plasmin was closely similar and in case of human plasmins averaged 29±0.9(SD) CTA units/mg plasmin and in case of canine plasmins 8.5±0.54(SD) CTA units/mg plasmin. Then, using human plasmin of the smallest size (mol wt 31,500), the total plasma antiplasmin capacity was determined in 20 normal human plasma, which averaged 7.8±2(SD) CTA units of plasmin per milliliter plasma. Studies were next made of the affinity of human [¹²⁵I]-plasmin of the smallest size with albumin, gamma globulin, α₂-macroglobulin, α₁-antitrypsin, fibrinogen, and fibrin. The results were 0%, 0%, 14.6±0.5(SD)%, 17.6±0.6(SD)%, 21±0.5(SD)%, and 20.5±0.6(SD)%, respectively, of [¹²⁵I]plasmin available and were unaltered when the amount of [¹²⁵I]plasmin was increased to twice and four times the original amount. Finally, the plasma disappearance half-life of canine [¹²⁵I]plasmin of the smallest size was studied in five healthy dogs, which averaged 14.2±0.63(SD) h. These results support the concept that the combination between plasmin and plasmin inhibitors is reversible and indicate that fibrinogen and fibrin have greater affinities than α₂-macroglobulin or α₁-antitrypsin.