AbstractIt has been proposed that buckling of actin stress fibers (SFs) may be associated with their disassembly. However, much of the detail remains unknown partly because the use of an elastic membrane sheet, conventionally necessary for inducing SF buckling with a mechanical compression to adherent cells, may limit high quality and quick imaging of the dynamic cellular events. Here, we present an alternate approach to induce buckling behavior of SFs on a readily observable glass plate. Actin SFs were extracted from cells, and constituent myosin II (MII) molecules were partially photo‐inactivated in contractility. An addition of Mg‐ATP allowed actin–myosin cross‐bridge cycling and resultant contraction of only thick SFs that still contained active MII in the large volume. Meanwhile, thin SFs with virtually no active motor protein in the small volume had no choice but to buckle with the shortening movement of nearby thick SFs functioning as a compression‐inducing element. This novel technique, thus allowing for selective inductions of contraction and buckling of SFs and measurements of the cellular prestress, may be applicable to not only investigations on their disassembly mechanisms but also to measurements of the relative thickness of individual SFs in each cell. J. Cell. Biochem. 113: 824–832, 2012. © 2011 Wiley Periodicals, Inc.