Tissue transglutaminase (tTGase) is a member of calcium-dependent transamidation enzyme family, but a detailed regulation mechanism of tTGase by intracellular Ca2+ is not clearly understood. Arachidonic acid (AA) and maitotoxin (MTX) activated tTGase in a dose- and time-dependent manner. Transfection of tTGase siRNA largely inhibited tTGase expression and tTGase activation by MTX. AA induced an initial increase of intracellular Ca2+ followed by a prolonged increase. Removal of extracellular Ca2+ with EGTA blocked the prolonged Ca2+ increase in response to AA, although the initial Ca2+ increase remained. In contrast, EGTA completely blocked the increase of intracellular Ca2+ by MTX. The activation of tTGase by AA or MTX was significantly inhibited by EGTA. Moreover, EGTA prevented the prolonged increase of intracellular Ca2+ and tTGase activation by lysophosphatidic acid, but had no effect on the initial Ca2+ increase. These results suggested that tTGase is regulated by the prolonged increase of intracellular Ca2+ originated from Ca2+ influx, rather than by the initial peak of transient Ca2+ increase. (c) 2005 Elsevier Inc. All rights reserved.