Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His 6 -tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mM ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.Homologous recombinational repair (HRR) 1 is a major DNA repair pathway that preserves chromosomal integrity during DNA replication and contributes to the removal of doublestrand breaks and interstrand cross-links from exogenous agents (see reviews in Refs. 1-3). HRR probably helps prevent double-strand breaks from arising during normal DNA replication and promotes their removal in an error-free manner when they do arise. In eukaryotic cells, this process is mediated by the highly conserved Rad51 DNA strand transferase and associated proteins that include distant relatives of Rad51, which are referred to as the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D). At least two stable complexes (a dimeric complex composed of XRCC3 and Rad51C and a larger complex composed of XRCC2, Rad51B, Rad51C, and Rad51D) have been found (4 -8). However, the exact role(s) these complexes play in HRR remains unclear. Mutations in the paralog genes lead to excessive spontaneous chromosomal aberrations and sensitivity to ionizing radiation (IR) and DNA cross-links (9 -13).Rad51 contains ATPase motifs composed of the Walker A and B boxes, and its DNA binding (14) and strand transferase activities (15, 16) are ATP-dependent. Structural analysis of archeal Rad5...