DNA fragmentation is a biochemical hallmark of apoptosis. Several endonucleases, including CAD/DFF40 and endonuclease G, are implicated in DNA fragmentation. DNase γ has also been considered to be one of the enzymes involved, but its role in relation to CAD/DFF40 in apoptosis has not been fully elucidated. Here, we distinguished between DNase γ-dependent and CAD/ DFF40-dependent DNA fragmentations. We found that DNase γ activities appeared in the late apoptotic phase and accelerated DNA fragmentation. Thus, even if the apoptotic DNA fragmentation is initiated by CAD/DFF40, DNase γ is required for the more complete digestion of the genomic DNA in dying cells.Apoptosis, or programmed cell death, eliminates unwanted cells during development, homeostasis, and immune responses (11,20). It is characterized by changes in cell morphology, including the formation of membrane-associated apoptotic bodies, membrane blebbing, nuclear breakdown, and chromatin condensation. At the molecular level, these changes are accompanied by the cleavage of several proteins and DNA fragmentation. DNA fragmentation is the final step in the disposal of the genomic DNA in cells undergoing apoptosis. The fragmentation of the genomic DNA in apoptotic cells occurs in two steps: the initial macrofragmentation (fragments ≧ 50 kb, a size consistent with that of a chromatin loop domain) and the subsequent internucleosomal DNA fragmentation, which is often observed as DNA laddering (12). The DNA fragmentation during apoptosis is not a passive process but rather an active process that participates in the enhancement of the apoptotic cascade in response to proapoptotic stimulations (2). However, the mechanism of the DNA fragmentation in apoptotic cells remains to be clarified. Although several endonucleases have been implicated in this process, their specific roles are still under debate. The DNA fragmentation factor (DFF) is a wellcharacterized apoptotic endonuclease (9). DFF is a heterodimer composed of a 40-kDa molecule and a 45-kDa molecule (DFF40 and DFF45, respectively).