Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.