In cerebellar granule cells, potassium cyanide (KCN) activates the NMDA receptor resulting in generation of nitric oxide and reactive oxygen species (ROS). To study the mechanism by which KCN stimulates ROS generation, the action of cyanide on the enzymatic pathways known to generate ROS were studied. The oxidant-sensitive fluorescent dye, 2,7-dichlorofluorescin was used to measure intracellular levels of nitric oxide and ROS in cerebellar granule cells. Using selective enzyme inhibitors, it was shown that both protein kinase C and phospholipase A2 are involved in KCN-stimulated generation of NO and ROS. In cells treated with indomethacin or nordihydroguairetic acid, inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX) respectively, attenuated (approximately 35%) KCN-induced generation of oxidant species. When L-NAME (LG-nitro-L-arginine methyl ester) (nitric oxide synthase inhibitor, NOS) was combined with either indomethacin or nordihydroguairetic acid, generation of oxidant species was blocked by more than 80%. Pretreatment with NS398 (COX-2 inhibitor) significantly decreased ROS generation indicating the involvement of COX-2 in KCN-induced oxidant generation. Treatment with L-NAME + NS398 blocked oxidant species generation, reflecting involvement of NOS. The participation of cytochrome P450 was not evident because SKF525A did not significantly reduce KCN-induced ROS generation. Furthermore, a correlation was observed between oxidant generation and lipid peroxidation of cellular membranes (as determined by thiobarbituric acid levels). Pretreatment with inhibitors of protein kinase C, phospholipase A2 or COX, LOX, COX-2 partially blocked KCN-induced formation of thiobarbituric acid reactive substance, whereas coincubation of L-NAME with the inhibitors decreased lipid peroxidation by 60 to 90%. In cytotoxicity studies, KCN-induced cell death was partially blocked by the inhibitors and significant protection was observed when L-NAME was combined with these compounds. These findings show that activation of phospholipase A2 and subsequent metabolism of arachidonic acid by the COX-2 and LOX pathways and NOS contribute to cyanide-induced ROS production.