The MAT alpha locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins, alpha 1 and alpha 2, which are responsible for determining the alpha-cell type. MAT alpha 1 is a positive regulator of alpha-cell-type-specific genes, and MAT alpha 2 is a negative regulator of a-cell-type-specific genes. MAT alpha 2 also determines the a/alpha diploid cell type, in conjunction with the MATa product, a1, by repressing haploid cell-type-specific genes. The MAT alpha 2-encoded protein binds specifically in vitro to a DNA sequence found upstream of several a-specific genes and is thus thought to exert its control directly at the transcriptional level of target genes. In an initial attempt to understand the molecular basis of the interaction of alpha 2 with DNA, we have saturated with missense mutations the segment of alpha 2 that is weakly homologous to a conserved prokaryote DNA-binding structure and to a portion of the higher eukaryote homoeo domain to ascertain the possible functional significance of this homology in alpha 2. We report here that most of the amino-acid residues in alpha 2 which correspond to conserved amino acids in the prokaryote DNA-binding proteins and in the homoeo domain are essential for the two repressor activities of alpha 2, that is, the repression of a-specific genes and of haploid-specific genes. Mutations in a subset of these amino-acid residues more severely affect the ability to repress a-specific genes than haploid-specific genes.