The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg(2+) or Mn(2+) and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans.