Toward the end of the 20th and beginning of the 21st centuries, clever in vitro biochemical complementation experiments and genetic screens from the laboratories of Xiaodong Wang, Shigekazu Nagata, and Ding Xue led to the discovery of two major apoptotic nucleases, termed DNA fragmentation factor (DFF) or caspase-activated DNase (CAD) and endonuclease G (Endo G). Both endonucleases attack chromatin to yield 3′-hydroxyl groups and 5′-phosphate residues, first at the level of 50–300 kb cleavage products and next at the level of internucleosomal DNA fragmentation, but these nucleases possess completely different cellular locations in normal cells and are regulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavage of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is regulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate folding during translation to generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducing factor (AIF). Understanding further regulatory check-points involved in safeguarding non-apoptotic cells against accidental activation of these nucleases remain as future challenges, as well as designing ways to selectively activate these nucleases in tumor cells. © 2005 Wiley-Liss, Inc.