Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[Ser]Sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[Ser]Sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[Ser]Sec following transfection of the mouse tRNA[Ser]Sec gene. The levels of selenocysteine tRNA[Ser]Sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[Ser]Sec gene. Although we were able to generate transfectants overproducing tRNA[Ser]Sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[Ser]Sec isoacceptors. In addition, increased levels of tRNA[Ser]Sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.