Coenzyme Q(10) (CoQ(10)) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. Last year, we reported the first mutations in CoQ(10) 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 CoQ(10) deficiency have not been well characterized. In this study, we investigated the consequence of severe CoQ(10) 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 CoQ(10) biosynthetic pathway produce different biochemical alterations. PDSS2 mutant fibroblasts have 12% CoQ(10) 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% CoQ(10) 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 CoQ(10) 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.