We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions. A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides. Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands. In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment. High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction. The synthetic Holliday junction was found to be a specific inhibitor of a S. cerevisiae enzyme that catalyzes the cleavage of Holliday junctions. Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1. Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex. The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays.