A glycolipid compound designated Macrophage Inhibitory Factor A3 (MIF-A3), isolated from M. avium serovar 2 (formerly M. paratuberculosis strain 18), has been shown to prevent the killing ofCandida albicans by activated bovine peripheral blood macrophages. Because both superoxide (O2-) and nitric oxide (NO) have been implicated in macrophage killing, it is possible that MIF-A3 could inhibit one or both of these systems. The present study tests this hypothesis. In a cell-free system, MIF-A3 significantly inhibited O2- production by xanthine-xanthine oxidase at 200, 400, and 800 mcg/ml concentrations [16.4±2.3%, 15.6±2.2%, and 17.0±3.1% respectively (mean ± SE, n=6-12,p < 0.05)], while the 100 mcg/ml concentration (7.5±3.0%) was not significantly different from a control glycolipid (5.7±2.7%). Similar results were obtained when hydrogen peroxide (H2O2) production was measured colorimetrically in sheep alveolar macrophages (2.0 x 106) stimulated with opsonized zymosan with or without pretreatment with various concentrations of MIF-A3 (100-400 mcg/ml) Zymosan stimulated alveolar macrophages produced 10.3±1.6 mcM H2O2, which was reduced to 5.5 mcM by the addition of 12,500 U/ml catalase. Stimulated alveolar macrophages pretreated with 200 and 400 mcg/ml MIF-A3 produced significantly less H2O2 [6.8±0.6 and 5.7±0.7 M, respectively (mean ± SE, n = 4-8, P < 0.050], while h2O2 production in alveolar macrophages pretreated with 100 mcg/ml was not significantly different (8.6±0.4 mcM). Phagocytosis as determined by counting zymosan particles within alveolar macrophages was unaffected. In addition, MIF-A3 caused a concentration dependent reduction of measurable H2O2 when incubated directly with exogenous H2O2. In contrast, NO production in murine C57BL/6 thioglycolate-elicited peritoneal macrophages (measured colorimetrically) after pretreatment with various concentrations of MIF-A3 (100-400 mcg/ml) and stimulation with lipopolysaccharide and -interferon was not significantly altered (n=3). These findings suggest that MIF-A3 acts as an oxygen radical scavenger which may be important in intracellular survival of mycobacteria.