The trypsin-inhibiting capacity of male adult mouse plasma was several-fold greater than that of the corresponding human plasma. Two major trypsin inhibitors were isolated from mouse plasma in an apparently homogeneous state. One seems to be homologous to alpha-1-antitrypsin isolated from several mammalian species, while the other, tentatively named contrapsin, does not correspond to any of the known plasma protease inhibitors that have been well characterized in human or other animals. When human plasma was fractionated in exactly the same fashion, no inhibitor corresponding to contrapsin was detected, suggesting the absence of this inhibitor in human plasma. No cross-reactivity was observed either between contrapsin and antiserum specific for alpha-1-antitrypsin or between alpha-1-antitrypsin and antiserum specific for contrapsin. alpha-1-Antitrypsin inhibited three serine proteases tested, i.e. porcine elastase, bovine trypsin, and chymotrypsin, whereas contrapsin inhibited only trypsin, but not the other two. The two inhibitors, however, shared a number of other properties in common. They were monomeric glycoproteins having total carbohydrate content of 9.6% in alpha-1-antitrypsin (Mr = 53,000) and 15.1% in contrapsin (Mr = 55,000). They had similar amino acid and carbohydrate compositions. They inhibited bovine beta-trypsin by forming stable complexes. They showed similar sex differences in blood concentrations. Serum levels of alpha-1-antitrypsin and contrapsin in males were 5.2 +/- 0.6 and 2.8 +/- 0.2 mg/ml, respectively, whereas those in females were 3.5 +/- 0.2 and 2.1 +/- 0.4 mg/ml, respectively.