This study evaluated the respiratory indexes (respiratory control ratio [RCR] and ADP/O ratio), mitochondrial transmembrane potential (ΔΨm), repolarization lag phase, repolarization level, ATP/ADP ratio, and induction of the permeability transition pore of brain mitochondria isolated from normal Wistar and GK diabetic rats of different ages (1.5, 12, and 24 months of age). The effect of amyloid β-peptides, 50 μmol/l Aβ_25–35 or 2 μmol/l Aβ_1–40, on mitochondrial function was also analyzed. Aging of diabetic mice induced a decrease in brain mitochondrial RCR, ADP/O, and ATP/ADP ratios but induced an increase in the repolarization lag phase. Brain mitochondria from older diabetic rats were more prone to the induction of the permeability transition pore, i.e., mitochondria from 24-month-old diabetic rats accumulated much less Ca²⁺ (20 μmol/l) than those isolated from 12-month-old rats (50 μmol/l) or 1.5-month-old rats (100 μmol/l). In the presence of 50 μmol/l Aβ_25–35 or 2 μmol/l Aβ_1–40, age-related mitochondrial effects were potentiated. These results indicate that diabetes-related mitochondrial dysfunction is exacerbated by aging and/or by the presence of neurotoxic agents such as amyloid β-peptides, supporting the idea that diabetes and aging are risk factors for the neurodegeneration induced by these peptides.