DNA fragmentation is a biochemical hallmark of apoptosis. Several endonucleases, including CAD/DFF40 and endonuclease G, are implicated in DNA fragmentation. DNase gamma 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 gamma-dependent and CAD/DFF40-dependent DNA fragmentations. We found that DNase gamma activities appeared in the late apoptotic phase and accelerated DNA fragmentation. Thus, even if the apoptotic DNA fragmentation is initiated by CAD/DFF40, DNase gamma is required for the more complete digestion of the genomic DNA in dying cells.