In Saccharomyces cerevisiae, of the many genes required for excision repair of ultraviolet-damaged DNA, only RAD1 and RAD10 also function in genetic recombination. Complex formation between the RAD1 and RAD10 gene products activates an endonucleolytic function that nicks single-stranded DNA and negatively supercoiled double-stranded DNA. To characterize the recombination role of the proteins Rad1 and Rad10, we have investigated their interaction with the Holliday junction, a four-stranded structure that results from single-stranded crossover between two duplex DNA molecules and whose resolution is obligatory for the generation of mature recombinants. We show that Rad1 binds specifically to a Holliday junction and, in the presence of magnesium, catalyses the endonucleolytic cleavage of the junction. Junction cleavage by Rad1 proceeds sufficiently without Rad10, thus identifying Rad1 as the catalytic subunit of Rad1/Rad10 endonuclease.