Factor B was isolated from fresh rat plasma by sequential chromatography on QAE-A50, Biorex-70, gel filtration on Sephadex G-200 superfine and rechromatography on QAE-A50. That Brat was isolated in its native form was indicated by its migration during immunoelectrophoresis and by its capacity to react with cobra venom factor (CoVF) in the presence of human D to form a C3 convertase capable of cleaving purified rat C3 and human C3. The recovery of Brat was between 8 and 15%; the final material was homogeneous according to SDS-PAGE analysis. Reduction of Brat with DTT in the presence of urea and SDS did not produce detectable peptides of lower molecular weight. Both reduced and unreduced Brat had an apparent molecular weight of 100,000. An antiserum against Brat induced in rabbits recognized only one protein in fresh rat plasma as indicated by immunodiffusion and immunoelectrophoretic analysis. Zymosan treatment of rat serum resulted in the cleavage of Brat into two fragments with alpha and gamma mobility. Native Brat has a beta electrophoretic mobility. The plasma concentration of Brat in Wistar rats was 215 +/- 38 microgram/ml (mean +/- SD).