Lipid vesicles containing phospholipids known to be present in substantial amounts in mitochondrial membranes were tested for their capacity to activate C1. Among them, only cardiolipin (CL) was highly efficient in C1 activation; no such effect was observed with phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. CL was shown to bind specifically C1q, because only unlabeled C1q competed with 125I-C1q for binding to CL. The requirement for C1q was confirmed by the finding that only fully reconstituted macromolecular C1, containing C1q, was activated by CL. The specificity of CL-induced activation of C1 was also demonstrated by introducing adriamycin, an agent known to interact with CL. Whereas adriamycin did not decrease C1 activation induced by immune complexes, it abrogated C1 activation by CL. The latter was shown to be a strong nonimmune activator of C1, because C1-INH did not inhibit CL-induced activation. When the concentration of CL in vesicles was decreased in the presence of phosphatidylcholine, C1 activation was detected only above a critical level of 35 mol% CL, compatible with a minimal density or clustering of CL molecules in the plane of the membrane. Moreover, C1 activation by CL was modulated by the addition of cholesterol. The threshold of CL required for C1 activation was lowered by the incorporation of more than 35 mol% cholesterol into the vesicles. These results show that CL incorporated into liposomes can be a potent nonimmune activator of C1. The negatively charged phosphate groups in CL are likely candidates for Clq-binding.