Coenzyme Q₁₀ (CoQ₁₀) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. Last year, we reported the first mutations in CoQ₁₀ biosynthetic genes, COQ2, which encodes 4-parahydroxybenzoate: polyprenyl transferase; and PDSS2, which encodes subunit 2 of decaprenyl diphosphate synthase. However, the pathogenic mechanisms of primary CoQ₁₀ deficiency have not been well characterized. In this study, we investigated the consequence of severe CoQ₁₀ deficiency on bioenergetics, oxidative stress, and antioxidant defenses in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations. Defects in the first two committed steps of the CoQ₁₀ biosynthetic pathway produce different biochemical alterations. PDSS2 mutant fibroblasts have 12% CoQ₁₀ relative to control cells and markedly reduced ATP synthesis, but do not show increased reactive oxygen species (ROS) production, signs of oxidative stress, or increased antioxidant defense markers. In contrast, COQ2 mutant fibroblasts have 30% CoQ₁₀ with partial defect in ATP synthesis, as well as significantly increased ROS production and oxidation of lipids and proteins. On the basis of a small number of cell lines, our results suggest that primary CoQ₁₀ deficiencies cause variable defects of ATP synthesis and oxidative stress, which may explain the different clinical features and may lead to more rational therapeutic strategies.—Quinzii, C. M., López, L. C., Von-Moltke, J., Naini, A., Krishna, S., Schuelke, M., Salviati, L., Navas, P., DiMauro, S., Hirano, M. Respiratory chain dysfunction and oxidative stress correlate with severity of primary CoQ₁₀ deficiency.