Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non‐homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA‐PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D‐NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B‐NHEJ). While D‐NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B‐NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70–80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D‐NHEJ proficient cells, or in cells where D‐NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D‐NHEJ or B‐NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4. J. Cell. Physiol. 213: 475–483, 2007. © 2007 Wiley‐Liss, Inc.