Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a site-specific double-stranded break at MAT, the yeast mating type locus. To identify other proteins involved in this process, we examined whether extracts prepared from ho- mutants contain additional factors that bind near the recognition sequence for Ho. Using an electrophoretic mobility shift assay, we isolated a chromatographic fraction that contains an activity, named YZbp, which binds to two sequences flanking the recognition sequence at MATalpha and to one sequence overlapping it at MATa. MAT plasmids carrying mutations in the YZbp recognition sequence are cleaved by purified Ho at wild-type efficiencies in an in vitro assay. These same plasmids, however, are not cleaved by Ho inside cells, demonstrating that YZbp acts as a positive activator of in vivo cleavage. YZbp is present in all cell types, even those not undergoing mating type switching, suggesting that it has additional cellular functions.