A protein was isolated from guinea-pig serum which enabled cobra venom factor (VF), in combination with purified C3 proactivator (C3PA) and Mg ++ , to induce turnover of isolated C3. To parallel the terminology for human serum, the protein was designated as factor D of the alternate pathway of complement activation. Purified factor D has a mol. wt of 22, 000 and an s-rate of 2·6. The isoelectric point was determined to be at pH 9·5. Factor D, although resistant to trypsin and hydrazine treatment, was relatively heat labile. It was found that the enzymatic activity directed against C3 was exerted by a trimolecular complex consisting of VF, C3PA and factor D. The existence of such a complex was demonstrated by gel filtration; additional evidence was provided by assembling an active complex on Sepharose-VF. Anti factor D serum blocked the C3 cleaving activity of the complex. Factor D itself was required for the C3 shunt activation induced by inulin, Dextran sulfate, Zymosan or endotoxin.