Death-associated protein kinase (DAP kinase) is a proapoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity.