The cytotoxic reactivity of 18 predefined class I HLA serum antibodies was compared with that of antibody preparations containing anticoagulants. ACD-A, EDTA, 4% citrate and heparin plasmas all showed lower cytotoxicity than serum antibodies. Recalcification of both platelet-rich and platelet-poor ACD-A plasma did not fully restore the antibody reactivity, suggesting a detrimental effect of calcium chelation. This effect was exclusive of volume or of any platelet or plasma protein involvement. The changes in pH contributed to the lower reactivity and to the increased lympholytic effect, whereas adjusting the pH toward the serum value improved the reactivity. Heat-inactivated antibodies showed only a slightly reduced cytotoxicity. Heparin had the least effect of all anticoagulants on the reactivity, although in heparin there was a definite dose-dependent decline in cytotoxic titer which was probably related to anticomplementary activity. Calcium chelators, such as EDTA and citrate, showed marked cytotoxic inhibition at half the usual complement concentration. This effect was more pronounced when the anticoagulant and lymphocytes were incubated prior to cytotoxicity testing. At the complement concentrations used, the inhibitory effects of the citrate anticoagulants appeared to be primarily calcium-related. Inhibition tests, serial titrations and testing of varying calcium concentrations confirmed the superiority of serum as antibody source.