An opsonic molecule designated the third component of hagfish complement (HC3), and a fragment of HC3 known as HC3b have recently been identified in the hagfish, Eptatretus burgeri. These proteins were purified from plasma and generated a set of several bands and/or smears during SDS-PAGE under standard, non-reducing conditions. Two-dimensional electrophoretic analysis of the proteins under non-reducing and reducing conditions revealed the breakdown of polypeptides at the site of a thioester bond and the concomitant partial release of a split product, depending on the weak covalent or non-covalent association of polypeptide chains, in a large fraction of molecules of HC3 during SDS-PAGE. Moreover, the heterogeneity of HC3b can be ascribed to the different configurations of subunits. A similar phenomenon was not observed in the case of lamprey C3, even though breakdown of polypeptides at a thioester bond did occur in some molecules.