Activation of human complement (C) occurred in vitro when mitochondrial membranes isolated from normal human heart tissue were incubated with normal human serum. This activation, as measured by C3 depletion, was not completely inhibited by blocking classical pathway activity in serum treated with EGTA, in C2-deficient serum, or in C1-depleted serum, nor in serum heated at 50 degrees C for 30 min to block the alternative pathway, but it could be prevented by blocking the classical and the alternative pathway simultaneously with EDTA, or by treating heated serum (50 degrees C. 30 min) with EGTA. Factor B was converted in normal serum as well as in EGTA-treated serum, but not in EDTA-treated serum. Mitochondrial membranes had no direct enzymatic or other activity that could inactivate functionally or highly purified C4 or C3, but the membranes could bind and activate C1 either in serum or in functionally pure C1 preparations. C4 also bound to the mitochondrial membranes only in the presence of C1. These data suggest that the activation of C by heart subcellular membranes involved both the classical and the alternative pathways, that the mitochondrial membrane preparations were capable of forming stabel complexes with C1 and C4, but not C3, and that the mitochondrial membrane preparations did not contain enzymes or have inherent properties that could directly cause C3 conversion.