Mutations in human mitochondrial tRNA genes cause a number of multisystemic disorders. A G-to-A transition at position 8313 (G8313A) transition in the mitochondrial tRNALys gene has been associated with a childhood syndrome characterized by gastrointestinal-system involvement and encephaloneuropathy. We have used transmitochondrial cybrid clones harbouring patient-derived mitochondrial DNA with the G8313A mutation for the study of the molecular pathogenesis. Our results showed that mutant mitochondrial cybrids respired poorly, and had severely defective mitochondrial protein synthesis and respiratory-chain-enzyme activity. Mutant cybrids also showed a marked decrease in tRNALys steady-state levels and aminoacylation, suggesting that these molecular abnormalities may underlie the pathogenesis of the mitochondrial G8313A mutation.