The homeodomain protein alpha2p plays a role both in transcriptional repression in the process of cell type determination and in donor selection during mating interconversion. We have explored the mechanism of alpha2p-directed donor selection by examining the effects on donor preference of mutants deficient in alpha2p-mediated transcriptional repression. As a transcriptional regulator, alpha2p interacts with Mcm1p, Tup1p, and Ssn6p to repress a-specific genes and with a1p, Tup1p, and Ssn6p to repress haploid-specific genes. We have found that mutant alleles of MATalpha2 that specifically diminish the interaction of alpha2p with Mcm1p or Tup1p behave as null alleles with regard to donor preference, while mutations of MATalpha2 that specifically diminish interaction of alpha2p with a1p behave as wild-type MATalpha2 in this capacity. Tup1p plays an essential role in alpha2p-mediated transcriptional repression, while Ssn6p has only a modest effect in repression. In a similar vein, we find that TUP1, but not SSN6, is required for proper donor selection. These results suggest that, in addition to regulating a-specific gene expression to establish the mating type of the cell, alpha2p-Mcm1p-Tup1p complex may indirectly regulate donor preference through transcriptional control of an a-specific gene. Alternatively, this complex may play a direct role in establishing donor preference via its DNA binding and chromatin organization capacity.