We present evidence for an activation step in the C1 molecule that occurs after the binding of C1 and before the appearance of C1̄ activity. Evidence for this internal activation step was obtained by the use of two different inhibitors of C1̄: C1̄-inactivator (I1) and a pentosan-poly-sulfo-ester (Sp54). Sp54 and I1 do not inhibit the uptake of C1. Sp54 inhibits a population of bound C1 not inhibitable by I1. C1 taken up in the presence of Sp54 are not inhibitable either by I1 or by Sp54. C1 taken up in the presence of I1 are inhibitable by Sp54 but not by I1. Sp54 is capable of inhibiting the hemolytic action of EAC1q4 while I1 is not. The data suggests that the internal activation step involves the subcomponent C1q.