tDNA sequences essential for promotion of transcription have been identified for a tRNA1met gene of X. laevis. A cloned tRNA gene unit was altered by resection of the 5' flanking sequences or by specific deletions of gene and trailing sequences. Gene internal sequences were also substituted by unrelated sequences. The gene units mutated in this way were coinjected into the oocyte together with cloned X. laevis 5S DNA, or were transcribed in vitro, in order to assess the effects of the sequence manipulation on transcription. We find major control sequences to be located near the 5' and the 3' ends of the sequences coding for the mature tRNA. A first such control sequence, having profound effects on the rate of tRNA production, has been mapped to sequence position 8-13 within the structural gene. A second regulatory sequence occurs within the region 51 to 72, that is, in or near the sequence coding for the pseudouridine loop of the tRNA. The sequences pinpointed in this way coincide with highly conserved sequences found in most, if not all, eucaryotic tRNAs. The anterior and the posterior control elements can be moved apart from one another without affecting the rate or points ot initiation and termination of transcription. While all deletions within the sequence coding for the mature tRNA led to inactivity of the mutated genes, substitution of the central portion by concatenated Hind III linkers produced gene units active in transcription. We postulate that the middle portion of the gene has a function in keeping the two control elements at sequence positions 8-30 and 51-72 at a critical distance from one another, a distance that can be enlarged but not shortened without obliterating the activity of the gene.