Mitochondrial DNA (mtDNA) depletion is associated with heterogeneous clinical phenotypes. The recent identification of the mutated genes in three groups of patients with mtDNA depletion had underscored the importance of the synthetic pathway of the mitochondrial nucleotides for mtDNA replication. Future goals include understanding how the defective proteins perturb replication, why it affects only some tissues and spares others, and which other genes should be considered in other patients with mtDNA depletion.