Rad51-mediated pairing between homologous DNA sequences during homologous recombination (HR) plays pivotal roles in DNA double-strand break repair. The multi-step process occurs through cooperation of Rad51 and a number of accessory protein factors. The development of various biochemical analyses with the requisite purified factors provides an opportunity to understand the molecular mechanisms of HR. In this chapter, we describe detailed procedures of in vitro assays using human Rad51, a polypeptide derived from the BRCA2 protein, and the Hop2–Mnd1 complex, to examine (1) homologous DNA pairing, (2) Rad51 targeting to single-stranded DNA, (3) stabilization of the Rad51 nucleoprotein filament, and (4) duplex capture by the Rad51 nucleoprotein filament. These methods are invaluable for delineating the functional interplay of HR factors.