Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at theMAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, α2p: Cells containing α2p select HMR, whereas those lacking α2p selectHML. As a repressor protein, α2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two α2p/Mcm1p-binding sites, DPS1 and DPS2,control donor selection. DPS1 and DPS2 are located ∼30 kb from the left arm of chromosome III, well removed fromHML, HMR, and MAT. Precise deletion of onlyDPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in α cells. Reciprocally, deletion of only the α2p binding segments in each of these two sites results in selection of the wrong donor loci in α cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of α2p, activates HML as donor. Binding of α2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML,resulting in default selection of HMR as donor. DPS1and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.