The triggering activities of heterologous fibrinogen and fibrin on endogenous production of tumor necrosis factor (TNF)-like cytotoxic factor in vivo were examined. The triggering activities of fibrinogen or fibrin from four species injected into the peritoneal cavity of C3H/He mice infected i.p. with bacillus Calmette-Guérin (BCG) were tested. Heterologous, but not homologous, fibrinogen and fibrin showed triggering activity. The route of triggering by heterologous fibrinogen to elicit TNF-like activity systemically was studied. Injection of heterologous fibrinogen i.v. into mice infected i.v. with BCG resulted in a 40-fold higher serum TNF-like activity level than after its i.p. injection. The serum TNF-like activity level was maximal 1 h after i.v. injection of heterologous fibrinogen. When heterologous fibrinogen was injected several times i.v. into mice bearing solid-tumors, TNF-like activity was also released into the serum after every injection, although the activity decreased progressively on second and third injections to 10 and 1%, respectively, of that after the first injection. We used heterologous fibrinogens derived from different species for triggering every week to avoid this gradual decrease of TNF-like activity. In this way TNF-like activity was induced as highly as the primary induction. These results showed that TNF-like cytotoxic factor could be produced in vivo locally or systemically by heterologous fibrinogen or fibrin. Thus both agents should be useful as nontoxic triggering agents.