The complement system is composed of 11 plasma proteins that, together with proteins of the properdin system, constitute an important humoral effector of immunological reactions (1). Several native serum complement (C) proteins have been shown to consist of two or more covalently bound polypeptide subunits. These include Clq (2), C3 (3), C4 (4), C5 (3), and C8 (1). Recently, a precursor form of the fourth component of guinea pig C was identified in studies of the cell-free synthesis of C4 and in homogenates of liver (5). The precursor of C4 (pro-C4) is a single polypeptide chain of mol wt 200,000, which is converted into native serum C4, a three chain protein of similar molecular weight (4, 6).The possibility was considered that other multichain C proteins were also synthesized as single chain precursors in analogy to pro-C4 and to other known precursor proteins such as proinsulin (7). In order to test this hypothesis, we analyzed the size and subunit composition of the third component of C (C3) synthesized in tissue culture and under cell-free conditions. Materials and MethodsPhenylmethylsulfonylfluoride (PMSF), 1 reduced glutathione, HEPES, GTP, creatine phosphokinase (CPK), phosphorylase A, and bovine serum albumin (BSA) were purchased from Sigma Chemical Co., St. Louis, Mo. ATP (dipotassium salt) and phosphocreatine (dipotassium salt) were from Calbiochem, La Jolla, Calif. Ribonuclease-free sucrose and ovalbumin (OVA) were from Schwartz/Mann Div., Becton, Dickinson and Co., Orangeburg, N. Y. Tritiated leucine, 45 Ci/ mmol, and a 14C-labeled I~amino acid mixture consisting of equal amounts of 14C-leucine (325 mCi/ retool), 14C-valine (251 mCi/mmol), 14C-isoleucine (321 mCi/mmol), and l~C-lysine (282 mCi/mmol) were obtained from New England Nuclear, Boston,, Medium 199 Special (lacking valine, leucine, isoleucine, lysine, and glutamine), and glutamine were purchased from Microbiological Associates, Bethesda, Md.; fl-galactosidase and carbonic anhydraso from Worthington Biochemical Corp., Freehold, N. J.; NCS solubilizer from Amersham/Searle Corp., Chicago, Ill.