The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38.0 daltons. Human C3bINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 ± 7 µg/ml (mean ± 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating β1H were removed from these proteins with anti-β1H Sepharose. However, in the presence of highly purified β1H and C3bINA, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and β1H had no effect on their structure. The action of C3bINA and β1H on C3b produced two fragments of the α'-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40.0 daltons. The action of C3bINA and β1H on C4b resulted in cleavage of the α'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments, which dissociated without reduction. To produce from C3b the immuno-chemically defined C3c and C3d fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.