Mutations in BCS1L, an assembly factor that facilitates the insertion of the catalytic Rieske Iron-Sulfur subunit into respiratory chain complex III, result in a wide variety of clinical phenotypes that range from the relatively mild Björnstad syndrome to the severe GRACILE syndrome. To better understand the pathophysiological consequences of such mutations, we studied fibroblasts from six complex III-deficient patients harboring mutations in the BCS1L gene. Cells from patients with the most severe clinical phenotypes exhibited slow growth rates in glucose medium, variable combined enzyme deficiencies, and assembly defects of respiratory chain complexes I, III, and IV, increased H(2)O(2) levels, unbalanced expression of the cellular antioxidant defenses, and apoptotic cell death. In addition, all patients showed cytosolic accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial networks, and decreased MFN2 protein levels. The observed structural alterations were independent of the respiratory chain function and ROS production. Our results provide new insights into the role of pathogenic BCS1L mutations in mitochondrial function and dynamics.