Barth syndrome (BTHS) is a multisystem disorder of individuals who carry mutations in tafazzin, a putative phospholipid acyltransferase. We investigated the hypothesis that BTHS is caused by specific impairment of the mitochondrial lipid metabolism. The fatty acid composition of all major mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), changed in lymphoblasts from BTHS patients. These changes were most extensive in CL and least extensive in PE. The complementary nature of the fatty acid alterations in CL and PC suggested that fatty acid transfer between these two lipids was inhibited in BTHS. Fluorescence staining and electron microscopy showed abnormal proliferation of mitochondria in BTHS lymphoblasts. The mitochondrial membrane potential, monitored with the fluorescence probe JC-1, was reduced in BTHS lymphoblasts. However, mitochondrial ATP formation of permeabilized lymphoblasts remained unaffected in BTHS. The data suggest that phospholipid abnormalities of BTHS mitochondria led to partial uncoupling of oxidative phosphorylation and that lymphoblasts compensated for this deficiency by expanding the mitochondrial compartment.