Human prothrombin fragments F1(αβ) and F2 were isolated as by-products of α-thrombin preparations. Prothrombin concentrates were activated to > 90% completion with thromboplastin (30–60 min, 23° C). The generated thrombin was quantitatively removed from clarified activation mixtures with Amberlite CG-50 resin (polymethylacrylic acid). The nonadsorbed fraction, containing the activation fragments, was desalted, freeze-dried, and chromatographed on DE-23 cellulose (diethylaminoethyl cellulose) employing an increasing salt gradient. Two pools containing F1(αβ) and F2 were obtained and further purified by gel filtration through Sephadex G-100. Fragment identity and purity were determined by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. The two isolated fractions migrated electrophoretically in SDS-containing gels as distinct nonreduced proteins ( M r 27,600 ± 1,300 and 15,800 ± 700 for F1(αβ) and F2, respectively). Following reduction, F2 remained electrophoretically homogeneous ( M r 15,800 ± 500). The reduced F1(αβ) gave rise to predominantly an M r 22,800 component and low M r materials (intact F1 was detected in some F1(αβ) preparations). Homogeneity of F2 was confirmed by detecting only an NH 2-terminal serine and a CO 2H-terminal arginine (1.1 mol. Arg/mol. F2 released by carboxypeptidase B digestion). Secondary cleavages within F1(αβ) were demonstrated by identifying threonine (0.44 mol. Thr/mol. Ala), and aspartic acid (0.15 mol. Asp/mol. Ala) as NH 2 terminal residues, and by obtaining arginine (0.6 mol. Arg/mol. FL(αβ) and trace amounts of lysine as the CO 2H-terminal residues). Sequential NH 2-terminal degradations (8 cycles) confirmed the deleted glycine at position-4 in human prothrombin and permitted identification of the internal F1 cleavage sites at Arg-52/Thr-53 and secondarily at Arg-55/Asp-56 (bovine prothrombin numbering). Since both cleavage sites are located within the same disulfide loops (Cys-48 to Cys-61), they account for the electrophoretic behavior of nonreduced versus reduced F1(αβ). Neither F1 (αβ) nor F2 exhibited biological activities similar to complement C3 or C5 fragments when examined in guinea pig ileal, skin permeability, or chemotaxis assays. The prothrombin fragments further did not promote cellular proliferation of cultured embryo fibroblasts. These findings suggest that the prothrombin fragments F1(αβ) and F2 are biologically inactive peptides except for their possible role of interfering with prothrombin activation.