During mating-type gene switching in Saccharomyces cerevisiae, DNA at the MAT locus is replaced by sequences copied from one of two unexpressed donor loci, HML or HMR, located near the two ends of the same chromosome and > or = 90 kb from MAT. MATa cells recombine nearly 90% of the time with HML, whereas MAT alpha cells select HMR. MATa donor preference was examined by deleting HML and inserting a donor at other chromosome III locations. MATa activated a large (> or = 40 kb) region near the left end of chromosome III, such that a donor placed at several sites within this domain was strongly preferred over HMR. When inserted outside of this domain, the donor was used equally with HMR. MATa donor preference for HML was abolished by the expression of the negative regulator, MAT alpha 2; however, HML regained its preferred status when the donor was unsilenced. Mating-type-dependent activation of the left end of the chromosome is also observed for other types of recombination that do not involve MAT switching. Spontaneous recombination between two leu2 alleles is 20-30 times higher in MATa than in MAT alpha when one of the leu2 alleles is inserted in place of HML. Transcription in this donor activation region is not affected by mating type. We conclude that MATa donor preference involves a mating-type-regulated change in the accessibility of a large chromosomal domain for recombination.