Group X secretory phospholipase A₂ (sPLA₂-X) has recently been shown to possess a powerful potency for releasing arachidonic acid from cell membrane phospholipids. Here, we report the purification of mouse pro- and mature forms of sPLA₂-X, as well as its expression and biological functions. Purified pro-sPLA₂-X was found to possess a propeptide of 11 amino acid residues attached at the NH₂-terminals of the mature protein, and showed as little as 8% of the PLA₂ activity of the mature form. Limited proteolysis of pro-sPLA₂-X with trypsin resulted in the appearance of the mature form with a concomitant increase in PLA₂ activity, suggesting a requirement of proteolytic removal of the propeptide for the optimal activity. The expression of sPLA₂-X mRNA was detected in various tissues including the lung, thymus, and spleen, and immunohistochemical analysis revealed its expression in splenic macrophages. In the spleen cells, mature sPLA₂-X elicited a prompt release of arachidonic acid with significant production of prostaglandin E₂ more efficiently than group IB and IIA sPLA₂s. In addition, sPLA₂-X was identified as a high-affinity ligand for both native and recombinant form of mouse PLA₂ receptor (PLA₂R). However, there was no significant difference in the sPLA₂-X-induced arachidonic acid release responses in the spleen cells between wild-type and PLA₂R-deficient mice. These findings strongly suggest that sPLA₂-X possesses two distinct biological functions in mice: it elicits a marked release of arachidonic acid from membrane phospholipids leading to the production of lipid mediators based on its enzymatic potency, and it acts as a natural ligand for the PLA₂R that has been shown to play a critical role in the production of inflammatory cytokines during endotoxic shock.