Some aspects influencing the serologic outcome of complement-dependent granulocyte cytotoxicity are presented. Fieoll-Hypnque (density 1.060) gradient centrifugation with hypotonic lysis of red blood cells yielded populations of granulmytes with greater than 90 per cent purity. Granulocytes exposed to papain (0.01%) at 24 C for 12 minutes showed enhanced serologic reactivity compared with untreated cells. In addition, enhanced cytotoxicity was promoted by 5 C (cold) as opposed to 22 C (warm) incubation of granulocytes and antibody in the first stage of the microcytotoxicity assay and emphasizes the temperature dependence of the seroiogic reactions. Neutrophil-specific leukoagglutinins were not cytotoxic under optimal in vifm conditions. Conversely, a dgnbkant proportiam of antibodies seh?cted for cytotoxicity failed to agglutinate granulocytes. Cold reactive granulocytotoxins are complement dependent, IgM in nature, and 2-mercaptaethanol sensitive and do not appear to be Immune complexes. By optimizing reaction conditions, granulocyte microcytotoxicity is a valuable addition to in vitro assays detecting immune sensitization against granulocyte surface antigens. THE NUMEROUS methodologies that describe the detection of granulocyte cell surface antigens testify as to the difficulties experienced with the present in vitro techniques. Such methods as immunofluorescence, antiglobulin consumption and polymorphonuclear function studies ,5*6*19~24*32 have definite limitations and are not suited to routine screening of antisera. To date, leukoagglutination assays18,20.26 have met with the most success in the definition of neutrophilspecific antigen groupsz1 although spontaneous aggregation of granulocytes and the need for functionally intact normal cells hamper this technique. Complement-dependent cytotoxic assays have utilized