The transcription of transfer RNA genes (tDNAs) and processing of the transcripts have been studied by injecting cloned tDNAs into Xenopus oocyte nuclei. Three main conclusions can be drawn. First, eucaryotic nuclear tRNA genes, but neither procaryotic nor mitochondrial tRNA genes, are expressed in injected oocytes. While both nematode and yeast tDNAS direct the synthesis of authentic tRNAs, neither E. coli tDNA nor human mitochondrial tDNAs support the synthesis of defined tRNAs when injected into oocytes. Second, competition experiments with co-injected 5S genes and inhibition experiments with alpha-amanitin show that injected tDNAs are transcribed by RNA polymerase III. Third, oocytes injected with a nematode tDNA synthesize a tRNA precursor which is processed post-transcriptionally by removal of a 5' leader sequence. This precursor is found exclusively in the nucleus and is processed in the nucleus before the mature tRNA enters the cytoplasm.