Background: DNA-PKcs stimulates the endonucleolytic activities of ARTEMIS but not the 5-exonuclease activity. Results: Point mutagenesis, small molecule inhibitors, and modulation of divalent cation concentrations all affect both the endonuclease and 5-exonuclease activities in parallel. Conclusion: ARTEMIS has intrinsic 5-exonuclease activity, in addition to its known endonuclease activity. Significance: ARTEMIS may use both its endo- and its 5-exonuclease activity in NHEJ. ARTEMIS is a member of the metallo--lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5- and 3-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5-exonuclease activity on single-stranded DNA and 5-overhangs, because this 5-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5-exonuclease activity. Third, divalent cation effects on the 5-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.