Abstract: We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLalpha or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML or HMR loci are largely identical in haploid a and a cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLalpha, unlike HMRa and most telomeres, shows increased NE-association in a strain lacking yKu70. Intriguingly, we find that the yeast Ku complex is associated with HML and HMR sequences in a mating-type specific manner. Its abundance decreases at the HMLalpha donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type specific binding of yKu to HMLalpha creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.