Several recent observations have indicated that the primary structure of the Chinese hamster selenocysteine tRNA([Ser]sec) is different than those of other mammalian species. These reports prompted us to investigate the gene sequence for this tRNA in Chinese hamsters. Southern blotting of Chinese hamster ovary (CHO) genomic DNA derived from cultured cells with a tRNA([Ser]sec) probe indicated several hybridizing bands, and each of the corresponding genetic loci was isolated from a recombinant CHO library by molecular cloning. Sequence analysis of these regions indicated three likely pseudogenes and a single functional gene whose sequence differed from those of other mammals. Of these, only one pseudogene and the putative functional gene are actively transcribed following their microinjection into Xenopus oocytes. The possibility that the functional CHO tRNA([Ser]sec) evolved from an edited transcript is discussed.