The only naturally occurring nonsense suppressor tRNAs described in higher eukaryotes are two opal suppressor serine tRNAs that occur in vertebrate tissues. These tRNAs have several unique features: (1) they are 90 nucleotides in length and thus are the longest tRNAs sequenced to date; (2) they are phosphorylated on their serine moiety to form phosphoseryl-tRNA; (3) they have few modified bases compared to other tRNAs; (4) they are encoded by a single gene even though several pyrimidine transitions occur post-transcriptionally and one of the transitions occurs in the anticodon; (5) the primary transcript arises, unlike any other known tRNAs, without processing on the 5' side of the gene product; and (6) the gene evolved more recently than other tRNA genes. An examination of the approximate origin and distribution of the opal phosphoserine gene in nature has shown that the gene occurs only on the Animal Kingdom and not in representatives of the Monera, Protist, Fungi and Plant Kingdoms. Among animals the gene occurs in members of the Phylum Chlordata (tunicate, amphioxus, lamprey, hag fish, horned shark, winter flounder, Xenopus, chicken and bovine), but not in members within the Phyla Arthropoda, Mollusca, Aschelminthes or Porifera. The genes encoding the opal suppressor tRNAs which have been isolated and sequenced from human, rabbit, chicken and Xenopus genomes are transcribed in vivo in Xenopus oocytes and are transcribed in vitro in HeLa cell extracts. Fingerprints of the processed transcript from the Xenopus gene show that the gene is faithfully transcribed and that initiation of transcription occurs at the first nucleotide within the gene.