To study oxidative mechanisms in cyanide toxicity, cyanide-induced generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescence dye 2,7-dichlorofluorescin. KCN produced a concentration-dependent (25-200 microM) generation of intracellular oxidant species that was blocked by N-methyl-D-aspartate receptor antagonists (MK-801 or AP5) or by removal of extracellular Ca++ from the incubation medium. To determine the relative contribution of NO and reactive oxygen species (ROS) to the increase of cellular fluorescence after KCN, a selective inhibitor of nitric oxide synthase, a NO scavenger and enzymes that metabolize ROS were added to the incubation medium. Interference with the nitric oxide system (reduced hemoglobin as a NO scavenger or [N(G)-nitro-L-arginine methyl ester [L-NAME] reduced fluorescence by 50%). Addition of enzymes that metabolize peroxide (catalase or superoxide dismutase [SOD]) also reduced fluorescence by nearly 50%. Combination of SOD with hemoglobin or L-NAME provided additional attenuation of the fluorescence and it was concluded that both NO and ROS are generated concurrently after KCN. Furthermore a correlation was observed between NO and ROS formation and levels of malonaldehyde (MDA), a marker of lipid peroxidation. Pretreatment with MK-801 blocked KCN-induced MDA formation, whereas L-NAME partially diminished MDA production. Treatment with a combination of SOD/catalase and L-NAME blocked the KCN-induced lipid peroxidation. In cytotoxicity studies cyanide-induced cell death was blocked by MK-801, whereas partial attenuation was produced by L-NAME; SOD/catalase treatments did not protect the cells. However, significant protection from cyanide-induced cytotoxicity was observed when L-NAME was combined with SOD/catalase. It is concluded that cyanide activates N-methyl-D-aspartate receptors to simultaneously generate both NO and ROS, which may lead to formation of the cytotoxic peroxynitrite anion.