Mitochondrial dysfunction is considered one of the pathophysiological mechanisms of autism spectrum disorder (ASD). However, previous studies of biomarkers associated with mitochondrial dysfunction in ASD have revealed inconsistent results. The objective of this study was to evaluate biochemical markers associated with mitochondrial dysfunction in subjects with ASD and their unaffected family members. Lactate and pyruvate levels, as well as the lactate-to-pyruvate ratio, were examined in the peripheral blood of probands with ASD (Affected Group, AG) and their unaffected family members (biological parents and unaffected siblings, Unaffected Group, UG). Lactate ≥22 mg/dl, pyruvate ≥1.4 mg/dl, and lactate-topyruvate ratio >25 were defined as abnormal. The clinical variables were compared between subjects with higher (>25) and lower (≤25) lactate-topyruvate ratios within the AG. The AG (n=59) had a significantly higher lactate and lactate-to-pyruvate ratio than the UG (n=136). The frequency of subjects with abnormally high lactate levels and lactate-to-pyruvate ratio was significantly higher in the AG (lactate 31.0% vs. 9.5%, ratio 25.9% vs. 7.3%, p<0.01). The relationship between lactate level and the repetitive behavior domain of the Autism Diagnostic Interview-Revised was statistically significant. These results suggest that biochemical markers related to mitochondrial dysfunction, especially higher lactate levels and lactateto- pyruvate ratio, might be associated with the pathophysiology of ASD. Further larger studies using unrelated individuals are needed to control for the possible effects of age and sex on chemical biomarker levels.