Activation of the alternative pathway of complement commences with the formation of an initial fluid-phase C3 convertase. Treatment of C3 with the nucleophilic reagent methylamine has previously been shown to result in the cleavage of an intramolecular thioester bond and to induce C3b-like properties, including the ability to form a fluid-phase C3 convertase. This report examines the hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway. The rate of spontaneous decay of C3 hemolytic activity in buffer was found to be between 0.2 and 0.4%/h. In the presence of other alternative pathway proteins, the rate of inactivation was 1%/h. The rate of spontaneous inactivation was greatly accelerated by low concentrations of chaotrophic agents such as KSCN or guanidine. Liberation of a sulfhydryl group, not present in native C3, correlated with loss of hemolytic activity, indicating that exposure to chaotropic agents resulted in thioester hydrolysis. Unlike native C3, C3 bearing a single reactive sulfhydryl group was capable of generating fluid-phase C3 convertase with Factors B, D, and P and was cleaved by Factor I (C3b inactivator) in the presence of Factor H (beta 1H). The fragmentation patterns indicated that the C3a domain was covalently associated with the functionally C3b-like C3. Organomercurial agarose was employed for the rapid removal of sulfhydryl-bearing, hemolytically inactive forms of C3 and C3b from native hemolytically active C3.