Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA(2)alpha involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA(2)alpha translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA(2)alpha inhibitors including pyrrophenone have been useful to understand cPLA(2)alpha function. Although this serine hydrolase inhibitor does not inhibit other PLA(2)s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1 er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA(2)alpha, C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA(2)alpha. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA(2)alpha function. (C) 2016 Elsevier Inc. All rights reserved.