The alternative oxidase (AOX) in plant mitochondria functions as a pathway to reduce reactive oxygen species (ROS) production. Transgenic tobacco cells with altered AOX levels were used to test this hypothesis. Antisense suppression of AOX increased ROS levels, while overexpression reduced them. Laser-scanning confocal microscopy showed that AOX affects mitochondrial-specific ROS formation. Antimycin A, which inhibits cytochrome electron transport, increased ROS production, and AOX overexpression reduced this effect. AOX overexpression also decreased the expression of ROS-scavenging enzymes like SOD and glutathione peroxidase, while increasing the expression of salicylic acid-binding catalase and pathogenesis-related protein (PR-1). These results suggest that AOX plays a role in lowering mitochondrial ROS formation in plant cells. ROS are produced during normal metabolic processes and can damage macromolecules if not removed. Mitochondria are a major source of ROS in eukaryotic cells, and excessive ROS production is linked to diseases in humans. In plants, mitochondria have a bifurcated electron-transport chain, with an alternative pathway that bypasses two of three sites where electron transport is coupled to ATP synthesis. AOX, a single protein in this pathway, is thought to exist as a homodimer in the inner mitochondrial membrane. AOX is encoded by a family of nuclear genes and is induced when the cytochrome pathway is inhibited, such as by antimycin A. This study used transgenic tobacco cells to measure ROS formation in intact cells, rather than isolated mitochondria, to better understand mitochondrial ROS production in vivo. The results showed that AOX levels inversely correlate with ROS levels. AOX overexpression reduced ROS production and the expression of ROS-scavenging enzymes, while AOX suppression increased ROS and the expression of ROS-sensitive genes. These findings support the hypothesis that AOX helps lower mitochondrial ROS formation in plant cells, which is important for plant resistance to stress conditions like chilling and pathogen attack. The study also highlights the role of AOX in plant defense mechanisms, as it is induced by pathogens and may be involved in resistance to viral infections. The findings suggest that AOX should be considered alongside other enzymes like SOD and catalase as a mechanism for plant protection against oxidative stress.The alternative oxidase (AOX) in plant mitochondria functions as a pathway to reduce reactive oxygen species (ROS) production. Transgenic tobacco cells with altered AOX levels were used to test this hypothesis. Antisense suppression of AOX increased ROS levels, while overexpression reduced them. Laser-scanning confocal microscopy showed that AOX affects mitochondrial-specific ROS formation. Antimycin A, which inhibits cytochrome electron transport, increased ROS production, and AOX overexpression reduced this effect. AOX overexpression also decreased the expression of ROS-scavenging enzymes like SOD and glutathione peroxidase, while increasing the expression of salicylic acid-binding catalase and pathogenesis-related protein (PR-1). These results suggest that AOX plays a role in lowering mitochondrial ROS formation in plant cells. ROS are produced during normal metabolic processes and can damage macromolecules if not removed. Mitochondria are a major source of ROS in eukaryotic cells, and excessive ROS production is linked to diseases in humans. In plants, mitochondria have a bifurcated electron-transport chain, with an alternative pathway that bypasses two of three sites where electron transport is coupled to ATP synthesis. AOX, a single protein in this pathway, is thought to exist as a homodimer in the inner mitochondrial membrane. AOX is encoded by a family of nuclear genes and is induced when the cytochrome pathway is inhibited, such as by antimycin A. This study used transgenic tobacco cells to measure ROS formation in intact cells, rather than isolated mitochondria, to better understand mitochondrial ROS production in vivo. The results showed that AOX levels inversely correlate with ROS levels. AOX overexpression reduced ROS production and the expression of ROS-scavenging enzymes, while AOX suppression increased ROS and the expression of ROS-sensitive genes. These findings support the hypothesis that AOX helps lower mitochondrial ROS formation in plant cells, which is important for plant resistance to stress conditions like chilling and pathogen attack. The study also highlights the role of AOX in plant defense mechanisms, as it is induced by pathogens and may be involved in resistance to viral infections. The findings suggest that AOX should be considered alongside other enzymes like SOD and catalase as a mechanism for plant protection against oxidative stress.