We have investigated whether arachidonic acid could regulate tissue transglutaminase (tTGase) via intracellular reactive oxygen species (ROS) in NIH3T3 cells. tTGase was identified in NIH3T3 cells by Western blot and confocal microscopy. Arachidonic acid elevated in situ tTGase activity in dose- and time-dependent manners with a maximal level at 1 h, and ROS scavengers, N-(2-mercaptopropionyl)glycine and catalase, blocked the tTGase activation by arachidonic acid. The activation of tTGase by arachidonic acid was largely inhibited by transfection of tTGase siRNA. The role of intracellular ROS in the activation of in situ tTGase was supported by the activation of in situ tTGase by exogenous H2O2. Arachidonic acid stimulated the formation of stress fibers in a dose- and time-dependent manner, and the ROS scavengers suppressed the arachidonic acid-induced formation of stress fibers. These results suggested that the activation of in situ tTGase and stress fiber formation by arachidonic acid was mediated by intracellular ROS in NIH3T3 cells. (C) 2004 Elsevier Inc. All rights reserved.