Activation of the alternative pathway of complement by immune complexes involves the covalent attachment of the third component (C3) to the IgG heavy chain. In order to localize the site/sites of attachment, adducts of human C3.IgG were digested in situ with endoproteinase Lys-C and Staphylococcus aureus V8 protease, and the fragments were analyzed. The dimeric peptide containing the covalent bond, identified by alkylation of the free thiol group (Cys1010) with iodo[14C]acetamide, was isolated by high-performance liquid chromatography fractionation. A double sequence with NH2 termini corresponding to position 134 of IgG heavy chain and position 1002 of the C3 alpha' chain was found by analysis with automated Edman degradation. The intact dimeric peptide had a mass of 3453 Da and was composed of IgG and C3 fragments with predicted sizes of 23 and 12 residues, respectively. The IgG peptide includes a cluster of six potential acceptor sites for ester bond formation. Thus, it appears that C3 binding is limited to a single region within the CH1 domain of the IgG1 heavy chain.