Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombre strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac4C-G-A-G-D-G-G-D-G-D-A-A-G-G-G-m22G-G-C-A-G-A-psi-U-U*-C-A-m1G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m5C-G-A-G-A-G-T-psi-C-G-m1A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-AOH. The anticodon sequence U*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.