Four related genes encode four different secretory phospholipase A2 (sPLA2) enzymes in mammals, namely the well described Group I and IIA enzymes and the more recently described Groups IIC and V. A large body of research has putatively demonstrated that the Group IIA sPLA2 is involved in diverse pathologic processes, such as rheumatoid arthritis, septic shock, intestinal neoplasia, and epidermal hyperplasia, as well as in cellular signaling by regulating the formation of arachidonate-derived lipid messengers. However, we demonstrate herein the involvement of another sPLA2, i.e. the Group V sPLA2, in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D1. Abundant message for Group V sPLA2 was detected in both resting and activated cells. In contrast, Group IIA sPLA2 message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA2 gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E2 production as well as reduction of the amount of sPLA2 protein at the cellular surface. Collectively, these results uncover Group V sPLA2 as a novel effector involved in arachidonic acid-mediated signal transduction.