We have previously shown gentamicin to form a redox-active iron chelate. This study investigates whether other aminoglycosides can likewise stimulate the generation of reactive oxygen species (free radicals). Kanamycin, neomycin and streptomycin were compared to gentamicin in intact cells and in cell-free in vitro assays using luminescence detection with lucigenin or luminol. Neutrophils and Epstein-Barr virus-transformed lymphoblastoid cells served as cell models in which a respiratory burst of superoxide was induced by phorbol ester. The addition of millimolar amounts of any of the aminoglycosides increased the luminescence significantly. The drugs also increased the formation of free radicals in an enzymatic (hypoxanthine-xanthine oxidase) and a non-enzymatic (phenazine methosulfate-NADH) superoxide-generating system. Half-maximal stimulation was reached with (0.4 mM gentamicin, and there was an absolute requirement for an electron donor, arachidonic acid. In both intact cells and cell-free systems, gentamicin-enhanced luminosity was suppressed by iron chelators. These results demonstrate that different aminoglycoside antibiotics can stimulate the formation of free radicals in biological and in cell-free systems. Luminescence detection is a convenient assay method to investigate the redox properties of these drugs.