In the present study, we characterized the generation of prostaglandin (PG)E₂ in human neutrophils. We found that the Ca²⁺-dependent type IV cytosolic phospholipase A₂ (cPLA₂) was pivotally involved in the COX-2-mediated generation of PGE₂ in response to a calcium ionophore, as determined by the use of selected PLA₂ inhibitors. PGE₂ biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA₂ activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE₂ production becomes favored over that of LTB₄ with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE₂ in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA₂ activation. Of the main eicosanoids produced by neutrophils, only LTB₄ was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE₂ biosynthesis in neutrophils.