Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.