We investigated the time course of ultrastructural changes of mitochondria in the spinal cord of homozygotes of Leu126TTdel SOD1 (superoxide dismutase 1) with FLAG (signal sequence at the C-terminal protein) transgenic mice (DF-homo). Non-Tg mice and wild-type human SOD1 with FLAG epitope transgenic mice (WF) were investigated as controls for non-onset Tg mice. Expansion and vacuolation of the mitochondrial matrix was exhibited in motor neurons in the anterior horns of DF-homo Tg mice at the presymptomatic stage. Such mitochondrial degeneration became severe at the postsymptomatic stage. In contrast, expansion of the mitochondrial inner-membrane space was not evident even at the terminal stage. Microvacuoles of cytoplasm and fibrillar inclusions were rarely shown from the early symptomatic stage. WF mice showed expansion and vacuolation of the mitochondrial inner membrane space at old age. Non-Tgs showed no obvious change in mitochondria. Gold-labeled human SOD1 immunoreactivity showed small amount of gold deposits in the vacuolated mitochondria. These results suggest that the expansion and vacuolation of mitochondrial matrix in the spinal cord of DF-homo transgenic mice is the first pathological change, but that it is not directly caused by the aggregation of an abnormal human SOD1 protein in intermembrane space of mitochondria.