The present studies describe a new function for calmodulin‐dependent protein kinase II (CaM‐KII) in signal transduction leading to apoptosis. Both tumor necrosis factor α (TNF) and UV light rapidly stimulated Ca²⁺‐independent activity of CaM‐KII in the monocytic leukemia, U937. Two mechanistically different inhibitors of CaM‐KII blocked activation of CaM‐KII and prevented DNA fragmentation and death. Activation of CaM‐KII during apoptosis and inhibition of DNA fragmentation by the two CaM‐KII inhibitors were reproduced in several other lines including KG1a, HL‐60, and YAC‐1. However, K562, which is relatively resistant to apoptosis induced by either TNF or UV light, did not activate CaM‐KII in response to these stimuli. A variant derived from U937 that is resistant to TNF‐ or UV light‐induced apoptosis also lacked a CaM‐KII response. Activation of Cam‐KII was blocked by two protease inhibitors, VAD‐fmk and TPCK, but not by other inhibitors of serine proteases. Both inhibitors of CaM‐KII and the protease inhibitors blocked activation of AP24, a serine protease originally isolated from apoptotic cells that induces DNA fragmentation in nuclei. Our evidence supports a model in which proteolytic activity functions upstream of CaM‐KII. This kinase then leads to activation of AP24, which transmits signals to the nucleus to initiate DNA fragmentation.—Wright, S. C., Schellenberger, U., Ji, L., Wang, H., Larrick, J. W. Calmodulin‐dependent protein kinase II mediates signal transduction in apoptosis. FASEB J. 11, 843–849 (1997)