Caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs during apoptosis. CAD is complexed with an inhibitor of CAD (ICAD) in non-apoptotic, growing cells. Here, we report that mouse WR19L and human Jurkat T lymphoma cells express two alternative forms of ICAD, ICAD-L and ICAD-S, at similar levels. CAD was predominantly associated with ICAD-L in these cell lines. When CAD was expressed alone in Sf9 cells, it was found in insoluble fractions. However, when CAD was co-expressed with ICAD-L and ICAD-S, it was recovered as a soluble protein complexed predominantly with ICAD-L. In vitro transcription and translation of CAD cDNA did not produce a functional protein. Addition of ICAD-L but not ICAD-S to the assay mixture resulted in the synthesis of functional CAD. These results indicated that ICAD-L but not ICAD-S works as a specific chaperone for CAD, facilitating its correct folding during synthesis. Recombinant CAD, as a complex with ICAD-L, was then produced in Sf9 cells. The complex was treated with caspase 3, and CAD was purified to homogeneity. The purified CAD had DNase activity with a high specific activity.