<p>SOD1 is the superoxide dismutase localized principally to the cytoplasm. It is found in all cell types, is extremely abundant, and highly efficient at converting superoxide (O₂^·−) to hydrogen peroxide (H₂O₂). SOD1 and its homologues in the mitochondrial matrix (SOD2) and extracellular environment (SOD3) are the only enzymes that act directly on superoxide. Surprisingly, <i>Sod1</i>^{<i>−/−</i>} knockout mice exhibit only a mild phenotype. In contrast, <i>Sod1</i>^{<i>−/−</i>} cells cannot survive and multiply in vitro. One possibility for the mild in vivo phenotype is that a subtle genome-wide mechanism of developmental compensation attenuates the phenotype. Conversely, many different mechanisms have been proposed to explain the lethality in vitro and the difficulties of rescuing it with antioxidants. Here, we aim to determine the cause of the severe in vitro phenotype of <i>Sod1</i>^{<i>−/−</i>} cells and whether the mild in vivo phenotype of <i>Sod1</i>^{<i>−/−</i>} mice reflects developmental compensation. We provide evidence that the lethality in vitro is directly due to superoxide toxicity and not to more indirect mechanisms. Furthermore, we created a floxed <i>Sod1</i> gene to compare acute adult-onset organism-wide removal of the protein with constitutive absence in mice and derived cells. We observed no phenotypic differences between acute and chronic loss of SOD1. We discuss how these observations can be reconciled with the evolutionary conservation of high levels of expression of a broadly expressed, high catalytic rate enzyme.</p>