To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.