Stress fibers (SFs) composed of nonmuscle actin and myosin II play critical roles in various cellular functions such as structural remodeling in response to changes in cell stress or strain. Previous studies report that SFs rapidly disassemble upon loss of tension caused by reduced myosin activity or sudden cell shortening, but the mechanism is unclear. Here, we showed that Rho-kinase inhibition with Y-27632 led to detachment of intact actin filaments from the SFs rather than depolymerization. Loss of tension may allow SFs to shorten via MgATP-driven cross-bridge cycling, thus we investigated the effects of MgATP concentration on SF shortening and stability. We performed the experiments using extracted SFs to allow control over MgATP concentration. SF contraction and disassembly rates each increased with increasing MgATP concentration. SFs transitioned from conventional SF shortening to rapid disassembly as MgATP concentration increases from 2 to 5 mM, which is within the physiological range of intracellular MgATP concentrations. Thus, we submit that SFs in intact cells are inherently on the verge of disassembly, which is likely due to the small number of actomyosin cross-bridges in SFs compared to those found in relatively stable myofibrils. Given that recent studies have revealed that loss of resistive force against myosin II could lower the fraction of the MgATPase cycle time that the myosin head is attached to actin (i.e., the duty ratio), binding of cytoplasmic levels of MgATP to myosin II may be sufficient to cause the disassembly of unloaded SFs. The present study thus describes a putative mechanism for rapid SF disassembly caused by decreased myosin activity or sudden cell shortening. (C) 2010 Elsevier Inc. All rights reserved.