Mitochondrial mutations are associated with a wide spectrum of human diseases. A common class of point mutations affects tRNA genes, and mutations in the tRNA-leu(UUR) gene (MTTL1) are the most frequently detected. In earlier studies, we showed that lung carcinoma cybrid cells containing high levels (greater than 95%) of mutated mtDNA from a patient with the pathological nucleotide pair (np) 3243 tRNA-leu(UUR) mutation can remain genotypically stable over time, and exhibit severe defects in mitochondrial respiratory metabolism. From such a cybrid containing 99% mutated mtDNA, we have isolated a spontaneous derivative that retains mutant mtDNA at this level but which has nevertheless reverted to the wild-type phenotype, based on studies of respiration, growth in selective media, mitochondrial protein synthesis and biogenesis of mitochondrial membrane complexes. The cells are heteroplasmic for a novel anticodon mutation in tRNA-leu(CUN) at np 12300, predicted to generate a suppressor tRNA capable of decoding UUR leucine codons. The suppressor mutation represents approximately 10% of the total mtDNA, but was undetectable in a muscle biopsy sample taken from the original patient or in the parental cybrid. These results indicate that the primary biochemical defect in cells with high levels of np 3243 mutated mtDNA is the inability to translate UUR leucine codons.