The proenzyme form of C1r was isolated by sequential chromatography of the euglobulin fraction of human serum on DE-32 cellulose, TEAE cellulose, and Sephadex G-200. C1r was found to be a proteolytic enzyme with β-globulin mobility, a sedimentation rate of 7.5S, and a molecular weight of approximately 188,000 daltons. Isolated C1r preparations exhibited an affinity for C1q and C1s, forming a reversible complex with C1q and a firm complex with C1s in free solution. Isolated C1r preparations did not activate proenzyme C1s, the natural substrate of the C1r enzyme, unless first activated by brief treatment with trypsin, a process accompanied by physicochemical changes in the molecule. It is not known whether the mechanism of conversion of C1r from a proenzyme into an active enzyme during C1 activation is also enzymatically mediated or, alternatively, whether conversion results from conformational changes in C1r induced by the attachment of C1, via C1q, to immunoglobulin molecules.