1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha′-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha′-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha′-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha′-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha′-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha′-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha′-chain. 5. C-Terminal analyses were done on the alpha′-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha′-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha′-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.

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