Résumé : It is now clear that the genetic code used in mitochondria differs in a number of ways from the standard code1-3, the most prominent being the use of the opal codon UGA to specify tryptophan4-7. This change in the mitochondrial genetic code is accommodated by the presence of an anticodon U*CA in mitochondrial (mt) tRNATrp from yeast8 and Neurospora crassa1. Furthermore, the translation of a mtDNA-encoded mRNA has been achieved in a eukaryotic cell-free system9: the mRNA of subunit II of yeast cytochrome c oxidase, which contains UGA codons in its reading frame, can be translated into a full-length protein if the system is supplemented with the Schizosaccharomyces pombe 10 cytoplasmic UGA suppressor tRNASer. It remains to be demonstrated whether a mitochondrial tRNA would be able to function in cytoplasmic protein synthesis. In the case of mt tRNATrp, this would cause the production of readthrough proteins due to the suppression of the UGA termination codon present in certain cytoplasmic mRNAs. Using the Xenopus oocyte microinjection system11-12, we show here that the mt tRNA Trp from Saccharomyces cerevisiae, when injected together with rabbit globin mRNA, suppresses UGA termination with high efficiency, thus leading to the production of a β-globin-related readthrough protein of molecular weight (Mr) 18,500. However, the suppressor activity of this tRNA within the oocyte cytoplasm is strictly dependent on the co-injection of an exogenous (Escherichia coli) acylating enzyme which is needed to charge the mt tRNATrp in vivo. The absence of an endogenous enzyme capable of acylating the yeast mt tRNATrp suggests that there is a biological barrier for the activity of a mt tRNA in the cytoplasm if a tRNA exchange between the two cellular compartments occurred. © 1981 Nature Publishing Group.