Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by beta-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using beta-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) beta-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms.