Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. Mutant SOD1 preferentially associates with the cytoplasmic face of mitochondria from spinal cords of rats and mice expressing SOD1 mutations. Two-dimensional gels and multidimensional liquid chromatography, in combination with tandem mass spectrometry, revealed 33 proteins that were increased and 21 proteins that were decreased in SOD1(G93A) rat spinal cord mitochondria compared with SOD1(WT) spinal cord mitochondria. Analysis of this group of proteins revealed a higher-than-expected proportion involved in complex I and protein import pathways. Direct import assays revealed a 30% decrease in protein import only in spinal cord mitochondria, despite an increase in the mitochondrial import components TOM20, TOM22, and TOM40. Recombinant SOD1(G93A) or SOD1(G85R), but not SOD1(WT) or a Parkinson's disease-causing, misfolded α-synuclein(E46K) mutant, decreased protein import by >50% in nontransgenic mitochondria from spinal cord, but not from liver. Thus, altered mitochondrial protein content accompanied by selective decreases in protein import into spinal cord mitochondria comprises part of the mitochondrial damage arising from mutant SOD1.