The formation of oxygen species during the redox cycling of sodium nitroprusside by rat liver microsomes and by chemical reductants was evaluated. The reduction of sodium nitroprusside by ascorbate and glutathione results in formation of the nitroprusside nitroxide radical which, on freezing at 77 K, results exclusively in the tetracyano [Fe(CN4)NO]2- and pentacyano [Fe(CN5)NO]3- forms of nitroxide radicals, respectively. The role of reducing agents on the inter-conversion of these two forms of nitroxide radical is discussed. The NADH and NADPH dependent microsomal reduction of nitroprusside results in the production of nitroprusside nitroxide radical, which in the presence of oxygen undergoes redox cycling to generate superoxide radical, and eventually hydroxyl radical is formed by a Fenton-type of reaction. Studies on the effect of several biologically or toxicologically relevant iron chelators on NADPH-dependent microsomal reduction of nitroprusside and subsequent formation of hydroxyl radical indicate that certain iron chelators such as isocitrate act as hydroxyl radical scavengers (depending on its concentration), but other chelators such as EDTA and DPTA function as good catalysts for the generation of hydroxyl radicals. The NADH and nitroprusside dependent microsomal production of hydroxyl radical is better in the presence of ATP, or equal in the presence of acetate, or diminished in the presence of DTPA when compared to the NADPH- and nitroprusside-dependent microsomal production of hydroxyl radicals. The effect of these chelates on the redox cycling of iron and nitroprusside by microsomes is discussed. Rat liver sub-mitochondrial particles and human hepatoblastoma cells (HepG2 cell line) also generated superoxide and hydroxyl radicals during the redox cycling of nitroprusside. These results provide direct evidence for the production of reactive oxygen species during the redox cycling of nitroprusside, The use of nitroprusside as a nitric oxide donor in biological systems may be complicated by the necessity to consider the generation of reactive oxygen species due to redox cycling of this compound by cellular reductases and low-molecular weight reductants.