The lack of direct proportionality between the number of SAC14 formed and the concentration of serum C1 was ascribed to the dissociation of C1 molecules into their subcomponents. We have obtained evidence, however, that this non-linear relationship is due to the dissociation from the C1 molecule of a hitherto unrecognized factor necessary for the activation of bound C1. Determination of the hemolytic activity of EAC14 before and after treatment with trypsin demonstrated that EAC14 contained two populations of SAC14, one in which C1 was activable by itself and by trypsin and another in which C1 was activable by trypsin but not by itself. A factor necessary for activation of C1, missing in non-self-activable C1, was designated “X” and the C1 molecules lacking this factor were designated (C1 - X). The finding that the apparent C1 content of whole serum was dependent on EAC4 concentration indicated that the activating factor “X” was bound independently from C1 and (C1 - X).