Human leukemia U937 cells are induced to undergo apoptosis by several chemotherapeutic agents; however, the cellular components involved in the process have not yet been identified. We found that an actin-cleavage activity (ACA) was activated in the VP-16-treated U937 cytosolic fraction and 15K- and 30K-actin fragments were produced. This ACA was inhibited by inhibitors of interleukin-1 beta-converting enzyme (ICE)/ced-3 family proteases, such as Z-Asp-CH2-DCB, YVAD-CHO, TPCK, TLCK, and iodoacetamide. Differing from ICE, the ACA could not process pro-IL-1 beta to mature IL-1 beta. Although ICE can cleave actin in vitro, ICE activity was not activated in the VP-16 treated U937 cells. These results indicate that actin is a potential substrate of ICE and ICE-like proteases, and that VP-16 preferentially activate an ICE-like protease, but not ICE itself, in U937 cells.