Apoptosis involves the proteolysis of specific cellular proteins by a group of cysteine proteases known as caspases. Many of these cellular targets are either functionally inactivated (e.g. poly(ADP-ribose) polymerase) or activated (e.g. other caspases, gelsolin) by such processing, thereby facilitating the cell death process. Caspase 3 is involved in the processing of many of these proteins. Recently, however, it was reported that caspase 3 is dispensable for the cleavage of a large number of cellular caspase substrates during apoptosis. Among these substrates is DFF-45/ICAD, a subunit of the heterodimeric DNA fragmentation factor (DFF), otherwise known as caspase-activated DNase (CAD), that mediates genomic DNA degradation during apoptosis. Conversely, others have reported that caspase 3 is essential for the cleavage and activation of DFF-45/ICAD. To resolve this controversy we examined DFF-45/ICAD processing during apoptosis in MCF-7 breast carcinoma cells that lack functional caspase 3 and in MCF-7 cells expressing caspase 3. We found that DFF-45/ICAD is cleaved by two distinct caspases, one of which is caspase 3. Furthermore, cleavage of the carboxyl-terminal region of DFF-45/ICAD, which is necessary for activation of the enzyme, requires functional caspase 3. In the absence of caspase 3 cleavage of the amino-terminal region of DFF-45/ICAD by another caspase occurs, but the DFF-45 enzyme remains inactive.