Selenocysteine is cotranslationally introduced into a growing polypeptide in response to certain UGA codons in selenoprotein mRNAs. The biosynthesis of this amino acid initiates by aminoacylation of specific tRNAs (designated tRNA([Ser]Sec)) with serine and subsequent conversion of the serine moiety to selenocysteine. The resulting selenocysteyl-tRNA then donates selenocysteine to protein. In most higher vertebrate cells and tissues examined, multiple selenocysteine isoacceptors have been described. Two of these have been determined to differ by only a single modified residue in the wobble position of the anticodon. In addition, the steady-state levels and relative distributions of these isoacceptors have been shown to be influenced by the presence of selenium. In order to gain a better understanding of the relationship between these tRNAs and how they are regulated, both the Xenopus selenocysteine tRNA gene and an in vitro synthesized RNA have each been injected into Xenopus oocytes and their maturation analyzed. In this system, selenium enhanced RNA stability and altered the distribution of isoacceptors that differ by a single ribose methylation. Interestingly, the biosynthesis of one of these modified nucleosides (5-methylcarboxymethyl-2'-O-methyluridine), which has been identified only in the wobble position of selenocysteine tRNA, also occurs in oocytes. Examination of the modified residues in both the naturally occurring Xenopus selenocysteine tRNA and the products generated from exogenous templates in oocytes demonstrated the faithful reconstruction of the biosynthetic pathway for these tRNAs.