Holliday junctions are central intermediates in the process of genetic recombination; they form as a consequence of a reciprocal exchange of strands between paired DNA molecules. Enzymes that specifically recognize and process these junctions are necessary for the formation of recombinant products. In the methods described here, we detail the in vitro construction of two types of Holliday junction: (1) a small synthetic junction formed by the annealing of partially complementary oligonucleotides; and (2) a true recombination intermediate structure formed by RecA protein-mediated strand exchange. The use of these substrates in assays designed to detect Holliday junction branch migration and resolution activities is described.