A method to obtain a highly purified Cls in the proesterase form is described for the first time. Neutral euglobulins were precipitated from fresh heparinized plasma in the presence of 0.06 M l-lysine, followed by extraction of Cls after suspending the euglobulin in 0.02 M EDTA, 0.005 M l-lysine (pH 7.5, relative salt concentration 0.04), and then DEAE cellulose column chromatography. The Cls preparation was shown to be homogeneous on immunoelectrophoresis developed against a potent anti-whole human serum and on analytical acrylamide gel electrophoresis. The yield of Cls was 10 to 20%. The Cls preparations have a specific hemolytic activity which is 20 to 30 times lower than C1s̅ and have a slower electrophoretic mobility on agar at pH 8.6. After incubating purified Cls with fractions containing Clr, Cls was activated as shown by an increase in its specific hemolytic activity and its faster migration to the anode on immunoelectrophoresis. The activating fractions were obtained from the DEAE column used to purify Cls and, although primarily Clr, they contain a few other proteins which do not bind at a relative salt concentration of 0.140. In addition to the different hemolytic activity and electrophoretic mobility, structural differences between Cls and C1s̅ were demonstrated by reduction and alkylation. Reduction of C1s̅ resulted in two major subunits with estimated molecular weights of 36,000 and 77,000. On the other hand, after reduction of Cls only one chain was found. Moreover, reduction of Cls which had been activated after purification resulted in the formation of two subunits similar to the subunits from C1s̅. The data indicate that activation is due to the cleavage of a peptide bond which converts a single polypeptide chain into two polypeptide chains linked by disulfide bonds. Experiments using insoluble C1s̅ (C1s̅ coupled to Sephadex G-25) showed that the destruction of C2 by C1s̅ occurred both in the presence of metals and in EDTA.