Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.