In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for use in oxygen-dependent killing mechanisms of invading pathogens.

Mutations in the gene coding for the NADPH oxidase cause an immunodeficiency syndrome called chronic granulomatous disease, characterized by extreme susceptibility to infection, especially catalase-positive organisms.

Genetic inactivation ("knockout") of SOD produces deleterious phenotypes in organisms ranging from bacteria to mice and have provided important clues as to the mechanisms of toxicity of superoxide in vivo.

Mice lacking mitochondrial SOD (MnSOD) die around 21 days after birth due to neurodegeneration, cardiomyopathy, and lactic acidosis.

[11] Mice lacking cytosolic SOD (CuZnSOD) are viable but suffer from multiple pathologies, including reduced lifespan, liver cancer, muscle atrophy, cataracts, thymic involution, haemolytic anemia, and a very rapid age-dependent decline in female fertility.

[11] Superoxide may contribute to the pathogenesis of many diseases (the evidence is particularly strong for radiation poisoning and hyperoxic injury), and perhaps also to aging via the oxidative damage that it inflicts on cells.

In model organisms (yeast, the fruit fly Drosophila, and mice), genetically knocking out CuZnSOD shortens lifespan and accelerates certain features of aging: (cataracts, muscle atrophy, macular degeneration, and thymic involution).

For practical purposes, this can be achieved only in vitro under non-physiological conditions, such as high pH (which slows the spontaneous dismutation) with the enzyme xanthine oxidase.

Researchers have developed a series of tool compounds termed "spin traps" that can react with superoxide, forming a meta-stable radical (half-life 1–15 minutes), which can be more readily detected by EPR.