Objectives: Several lines of evidence suggest that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis (ALS), but despite the fact that mitochondria play a central role in excitotoxicity, oxidative stress, and apoptosis, the intimate underlying mechanism linking mitochondrial defects to motor neuron degeneration in ALS still remains elusive. This study was performed to assess the mitochondrial respiratory chain dysfunction and cellular energy index (ATP/ADP ratio) in lymphocytes of ALS patients. Methods: In this study, activity of mitochondrial respiratory chain complex I (measured as NADH-ferricyanide reductase) and both intracellular ATP and ADP measurements were performed on lymphocytes of ALS patients (n = 14) and control subjects (n = 26). Then, ATP/ADP ratio was calculated. Results: Our finding showed that in patients compared with controls, complex I activity and intracellular ATP were significantly reduced (P = 0·001) and intracellular ADP content was increased (P<0·005) and ATP/ADP ratio subsequently was decreased and also we found strong correlation between complex I activity and intracellular ATP content and strong reverse correlation between complex I activity and intracellular ADP content in the patients with ALS (r² = 0·90). Discussion: This study suggests that complex I deficiency and both reduction in intracellular ATP and increase in intracellular ADP content may be involved in the progression and pathogenesis of ALS.