Synthesis of hemolytically active C2 was detected in short-term tissue cultures of guinea pig spleen and to a lesser extent in cultures of lung, lymph nodes and cells obtained from bone marrow and peritoneal exudates. The in vitro production of C2 by these tissues was highly temperature-dependent and was reversibly inhibited by puromycin and cycloheximide. Fragments of spleen incorporated 14C-amino acids into a molecule that was immunochemically and functionally identical with C2. In vitro production of C2 was not detected in short-term cultures of kidney, skin, muscle, intestinal tract and liver.