In the nonhomologous end joining pathway of DNA double-strand break repair, the ligation step is catalyzed by a complex of XRCC4 and DNA ligase IV. Extracts of CHO-K1 cells are able to accurately rejoin a site-specific free radical-mediated double-strand break with partially complementary overhangs, by a mechanism involving alignment-based gap filling followed by ligation. Extracts of XR-1 cells, which lack XRCC4 and DNA ligase IV, carried out neither gap filling nor ligation. Supplementation of the extracts with recombinant XRCC4/ligase IV, but not with XRCC4 alone, restored gap filling and accurate end joining. The results imply that XRCC4 and ligase IV are essential for alignment-based gap filling, as well as for final ligation of the breaks.