Immunoprecipitates of human C4 from EDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the α-chains of the secreted (C4(s)) and predominant plasma (C4(p)) forms of C4, two additional molecules with apparent molecular weights of ~168,000 (p168) and ~125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of ~5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or α2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C4(s) and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14 ± 4.8 and 21 ± 9.2% (mean ± SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4(s). Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the β- and α-chains and between p125 and the α- and γ-chains. Partial NH2-terminal sequencing revealed that the β-chain was NH2-terminal in p168 and that the α-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent uncleaved β-α- and α-γ-fragmented pro-C4, respectively. Thus, in most individuals, plasma C4 consists of five structurally distinct molecules, the single polypeptide precursor (pro-C4), the three-subunit secreted (C4(s)) and predominant plasma (C4(p)) forms of C4, and two incompletely processed two-subunit molecules with uncleaved β-α- (p168) or uncleaved α-γ- (p125)-subunits. In addition, all five molecules are observed for both C4A (Rodgers) and C4B (Chido) structural genes.