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Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson's disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP + (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP + stimulates ROS production by raising cytoplasmic Ca 2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca 2+ , MPP + co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other's function. (3) Ca 2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn 2+ . (4) Zn 2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca 2+ and Zn 2+ , and prevention of electron escape from complexes -all abolished the ability of MPP + to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP + , while Zn 2+ ionophore replicated the effects of both MPP + and Ca 2+ . Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca 2+ , Zn 2+ , and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases., (© 2024. The Author(s).)