Transcription of Drosophila tRNA genes is controlled by signals within and outside the region coding for the mature tRNA. Deletion analysis has revealed an oligonucleotide sequence in the 5'-flanking region of a Drosophila tRNA2Lys gene to be responsible for the poor transcriptional activity of this and of other tRNA genes. The low template activity of this gene was maintained even after deletion of the 5'-flanking region up to nucleotide -23. Removal of nine additional nucleotides resulted in complete loss of transcriptional repression. The oligonucleotide responsible for transcriptional repression is GGCAGTTTTTG and is located 13 nucleotides upstream from the mature tRNA coding sequence. Since the sequence of the undecanucleotide is well conserved within the 5'-flanking region of all known Drosophila tRNA2Lys genes, we have investigated why the transcription of all these genes is not similarly repressed. Deletion or insertion of nucleotides between the mature tRNA coding region and this oligonucleotide resulted in tRNA genes with increased template activity. This observation suggests that the position of this oligonucleotide relative to some element downstream influences the extent of transcriptional repression.