The plasmin inhibiting activity was determined in gel filtration fractions of human serum containing [alpha]2-macroglobulin. The ratio between the plasmin inhibition and [alpha]2-macroglobulin concentration in 5 fractions was approximately constant and was about 1 mole of plasmin per mole of [alpha]2-macroglobulin. The ratio between the trypsin-binding and the plasmin-binding activity (both expressed in mole/ml) of a purified [alpha]2-macroglobulin preparation was determined as 1.80. The binding of plasmin to [alpha]2-macroglobu-lin blocked about half of its trypsin binding capacity. The findings suggest that 1 mole of [alpha]2-macroglobulin binds 1 mole of plasmin and 2 moles of trypsin and that plasmin can be bound to 1 of the trypsin binding groups of the [alpha]2-macroglobulin.