Two different allele-specific suppressor mutants of the fission yeast Schizosaccharomyces pombe produce opal (UGA) suppressor tRNAs. This was shown by the use of a new in vitro assay for eukaryotic nonsense suppression: a wheat germ extract is programmed with rabbit globin mRNAs and the readthrough products are studied. alpha-Globin is elongated upon addition of ochre (UAA) suppressor tRNAs, whereas beta-globin yields a readthrough product with opal suppressor tRNAs. This simple and very sensitive assay allowed the purification of the opal suppressor tRNA from S. pombe strain sup3-e. The pure tRNA can be aminoacylated with serine; thus, we conclude that this suppressor tRNA inserts serine in response to the UGA termination codon of pure rabbit beta-globin mRNA.