1. The evidence presented indicates that Mg++, or other cation such as Ca++, Ni++, or Co++, is essential for the hemolytic action of C'. Ca++, Ni++, and Co++ are less effective than Mg++. The hemolytic system usually does not contain sufficient Mg++ for optimal hemolytic activity so that a marked enhancement can be obtained by addition of extra Mg++. 2. The enhancing action of tissue fluids can be ascribed to their contribution of Mg++. 3. Substances which bind Mg++ and Ca++ are anticomplementary when added to the usual hemolytic system which contains only a small quantity of Mg++. This type of anticomplementary effect can be overcome by addition of extra Mg++. 4. Ca++ may also be essential to the lytic process but its action is much less pronounced than that of Mg++.

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One approach to the problem of the biological function of complement is the comparative study of this strange and unstable complex as it occurs in the sera of different species of animals. The first of two recent studies along these lines was that of Hegediis and Greiner (1). The reasonable hypothesis was propounded that the titer of a complement is limited by the component present in lowest titer, and that each of the four components might be titrated independently by addition of the other three components in excess, so as to make the component in question the one present in lowest titer. Required reagents were prepared by standard methods from guinea pig complement, apparently in the firm conviction that each component of this complement was wholly equivalent in function to the corresponding component of the complement of the animal species being tested, and vice versa. The validity of this assumption was denied by Ecker, Pillemer, and Seifter in a comparative study of human and guinea pig complements (2) because certain substitutions were not found "effective."The complements of these two species have been under investigation for several years in the laboratories of the Presbyterian Hospital (3-8, and unpublished studies). While there had been no reason to question the assumptions underlying the work of Hegediis and Greiner, doubts had arisen as to the adequacy of their technique for measuring the various components and their conclusions as to the relative quantities of the components in guinea pig complement had not been confirmed. It also became evident that experimental conditions could be found under which the components of human and guinea pig complements are mutually substitutive 1 and that existing methods for the titration of the components of complement required revision in order to render them suitbd to their purpose. It was therefore decided to submit evidence on these matters already in hand and to extend the study in order to learn more

Experiments are described indicating that the magnitude and sensitivity of the passive cutaneous anaphylaxis (PCA) response in normal rats to a given level of immune reagents, may be enhanced by the addition of hemolytically active sera. A similar enhancement in normal rats has been obtained with C' component reagents possessing properties associated with the third component of C'. Parallelisms between in vitro fixation of C' and PCA induction by antigen and antibody are shown. The horse anti-pneumococcus system has low C'-fixing potencies and is also less efficient than the rabbit polysaccharide system in the induction of PCA. Findings of a similar nature were observed in the reaction of rabbit anti-ribonuclease with ribonuclease, the acetylated and guanidinated derivatives of the enzyme. The injection of hemolytically active serum into C'-deficient rats was accompanied by a partial restoration of PCA. Restorative effects were also noted with heated and ammonia-treated serum. The return of hemolytic potency and responsiveness to PCA in C'-depleted rats, follow a similar time course. The data presented indicate that the PCA reaction can be studied as a function of at least three variables, antigen, antibody, and a serum constituent resembling C'.

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