Sequence analysis of a Schizosaccharomyces pombe DNA fragment revealed two tRNA coding regions separated by a seven nucleotide spacer. the 5'-proximal tRNA gene encodes a tRNAUCGSer sequence, which is interrupted by a 16 nucleotide intron at the 3' side of the base adjacent to the anticodon. The second tRNA gene encodes an initiator tRNAMet sequence. This DNA fragment, cloned into pBR322, was used as template for in vitro transcription in a nuclear extract of Xenopus oocytes. The tRNA genes were transcribed into one RNA precursor which contained both tRNA sequences. The primary transcription product initiates with pppG, contains a 9 nucleotide leader sequence, a 16 nucleotide intron, a 7 nucleotide spacer between the two tRNA molecules and an 8--9 nucleotide trailer sequence. RNA initiation was only observed upstream of the 5'-proximal tRNASer. We used RNA analysis to establish a sequence of the enzymatic steps of tRNA maturation in the nuclear extract. The first step in processing the dimeric precursor is an endonuclease cleavage which generates the mature 5' end of the tRNAMet. Further steps include the removal of the flanking sequences and addition of the CCAOH 3' terminus. The last step is the splicing of the tRNASer precursor to remove the intervening sequence.