Previous work showed that in human nuclear extracts, double-strand break substrates bearing partially complementary (-ACG) 3′-phosphoglycolate (PG)-terminated 3′ overhangs are joined by a mechanism involving annealing of the terminal CG dinucleotides, PG removal, single-base gap filling and ligation. However, in these extracts only a minority of the breaks are rejoined, and most of the 3′-PG termini remain intact even after several hours. To determine whether the presence of a persistent 3′-PG prevents patching and ligation of the opposite strand, a substrate was constructed with two -ACG overhangs, one PG-terminated and one hydroxyl-terminated. after incubation in HeLa cell nuclear extracts, two major repair products of similar yield were formed: a fully repaired duplex and a nicked duplex in which the initial 3′-PG terminus remained intact. These results indicate that patching and ligation can proceed to completion in the unmodified strand despite persistence of the 3′-PG-terminated break in the opposite strand. The break in the PG-containing strand could then presumably be rejoined by a single-strand break repair pathway.