We studied the cellular distribution of both deleted (Δ) and wild-type (wt) mitochondrial DNAs (mtDNAs) in ‘normal’ and respiration-deficlent muscle fibers from four patients with mitochondrial myopathy. PCR-based methods were used to quantitate both relative and absolute amounts of Δ-and wt-mtDNAs in microdissected fiber segments. Although Δ-mtDNAs were present in normal fibers (31% ± 26), their percentages were much higher in affected fibers (95% ± 2). Absolute levels of Δ-mtDNA were also increased in affected fibers, whereas levels of wt-mtDNA were significantly reduced in these fibers. These results Indicate that a threshold ratio of Δ-/wt-mtDNA must be achieved before an impairment of respiration is observed in muscle. Moreover, the marked reduction In wt-mtDNA observed in affected fibers suggests that absolute amounts of mtDNA may play a role in the pathogenesis of mitochondrial myopathies.