Cytoskeletal proteins are key participants in the cellular progression to apoptosis. Our previous work demonstrated the critical dependence of actomyosin rearrangement and MLC phosphorylation in TNF‐α‐induced endothelial cell apoptosis. As these events reflect the activation of the multifunctional endothelial cell (EC) MLCK isoform, we assessed the direct role of EC MLCK in the regulation of TNF‐α‐induced apoptosis. Bovine pulmonary artery endothelial cells expressing either an adenovirus encoding antisense MLCK cDNA (Ad.GFP‐AS MLCK) or a dominant/negative EC MLCK construct (EC MLCK‐ATP‐del) resulted in marked reductions in MLCK activity and TNF‐α‐mediated apoptosis. In contrast, a constitutively active EC MLCK lacking the carboxyl‐terminal autoinhibitory domains (EC MLCK‐1745) markedly enhanced the apoptotic response to TNF‐α. Immunostain‐ing in GFP‐EC MLCK‐expressing cells revealed colocalization of caspase 8 and EC MLCK along actin stress fibers after TNF‐α. TNF‐α induced the caspase‐dependent cleavage of EC MLCK‐1745 in transfected endothelial cells, which was confirmed by mass spectroscopy with in vitro cleavage by caspase 3 at LKKD (D¹⁷⁰³). The resulting MLCK fragments displayed significant calmodulin‐independent kinase activity. These studies convincingly demonstrate that novel interactions between the apoptotic machinery and EC MLCK exist that regulate the endothelial contractile apparatus in TNF‐α‐induced apoptosis.—Petrache, I., Birukov, K., Zaiman, A. L., Crow, M. T., Deng, H., Wadgaonkar, R., Romer, L. H., Garcia, J. G. N. Caspase‐dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF‐α‐mediated bovine pulmonary endothelial cell apoptosis. FASEB J. 17, 407–416 (2003)