A three-base substitution mutant of the yeast tRNALeu3 has been constructed. The mutation, introduced through the use of a heptadecanucleotide as a site-specific mutagen, is localized in the anterior portion of the promoter and results in the inability to form a D stem. The mutant is active in transcription, but maturation of the 5' terminus and splicing are abolished. The results are discussed in the light of a recently proposed model for initiation of transcription of eucaryotic tRNA genes.