A modified cytotoxicity assay was adapted from classical erythrocytolytic assays, in which complement components were added in sequence to antibody-sensitized cells.This assay was applied to a model system in which mouse sarcoma cells were sensitized with H-2 alloantibody. The stepwise presentation of complement components combined with the stabilization of C2 by iodine treatment considerably augmented the lytic efficiency of human complement. More generally, the techniques adopted for this study provide a new model for obtaining basic information about selective reaction steps concerned in lysis of nucleated cells by alloantibody and complement.Comparisons of the lysis of sheep erythrocytes by xenoantibody with the lysis of mouse sarcoma cells by H-2 alloantibody, in our assay system with oxidized or untreated human complement, disclosed a difference in the kinetics of C142 formation, manifest in a lag phase and a protracted tmaxfor the sarcoma cells. These data may suggest that multiple complement-mediated functional lesions are necessary for immune lysis of nucleated cells.Selective lysis of nucleated cells by antibody and complement has become an indispensable tool for investigating cell surface antigens that characterize sets of lymphocytes with distinct functions(1). The delineation and analysis of several systems of cell surface antigens, such as the Lyt(2, 3)and Lyb(4, 5)series, depends critically on the sensitivity of the cytotoxic assay.Repeatedly, in the past, improvements of complementation in the cytotoxicity assay have come from a better understanding of the factors involved in the complement-mediated lysis of nucleated cells(6). There is abundant information on mechanisms of complement activation and function in hemolytic systems (7) but much less is known of the role of complement in the lysis of nucleated cells(8–14). In particular, no comparative study on reaction sequence and contribution of isolated complement components for nucleated cell lysis is so far available. We show here that lysis of nucleated cells by H-2 antibody can be markedly increased by the use of chemically treated complement(15)in an appropriately modified cytotoxicity assay, presenting complement components in sequence. In addition, we present new basic information about sequential steps of complement binding and activation as these affect nucleated cell lysis in comparison with the classical reaction sequence in erythrocytolysis.