MITOCHONDRIAL encephalomyopathies are usually divided into three distinct clinical subgroups: (1) mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS); (2) myoclonus epilepsy associated with ragged-red fibres (MERRF); and (3) chronic progressive external ophthalmoplegia (CPEO) including Kearns-Sayre syndrome^1–5. Large deletions of human mitochondrial DNA and a transition mutation at the mitochondrial transfer RNA^lys gene give rise to CPEO including Kearns–Sayre syndrome^6–8 and MERRF^9,10, respectively. Here we report an A-to-G transition mutation at nucleotide pair 3,243 in the dihydrouridine loop of mitochondrial tRNA^Leu(UUR) that is specific to patients with MELAS. Because this mutation creates an Apal restriction site, we could perform a simple molecular diagnostic test for the disease. The mutation was present in 26 out of 31 independent MELAS patients and 1 out of 29 CPEO patients, but absent in the 5 MERRF and 50 controls tested. Southern blot analysis confirmed that the mutant DNA always coexists with the wild-type DNA (heteroplasmy).