Cytosolic phospholipases A2 (cPLA2) and cyclooxygenases-1 and -2 (COX-1 and -2) play a pivotal role in the metabolism of arachidonic acid (AA) and in eicosanoid production. The coordinate regulation and expression of these enzymes is not well defined. In this study, the effect of phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNFalpha), lipopolysaccharide (LPS) and macrophage-colony stimulating factor (M-CSF) on AA release and prostaglandin E2 (PGE2) production and the expression of cPLA2 and COX-1 and -2 were investigated in U937 human pre-monocytic cells and fully differentiated macrophages. Treatment of U937 cells with PMA or macrophages with LPS increased AA release and PGE2 production. Incubation of U937 cells or macrophages for 8 h with all stimuli elevated cPLA2 expression. In contrast, cPLA2 expression was reduced upon further incubation of U937 cells or macrophages for 24 h with all stimuli indicating a bi-phasic expression pattern of this enzyme. PMA induced COX-1 expression in U937 cells whereas LPS induced COX-2 expression in macrophages. Although TNFalpha and M-CSF induced a significant amount of AA release in both cell models, they failed to induce a comparable production of PGE2 since they were unable to induce the coordinate expression of the downstream key enzymes, COX-1 or COX-2. The results suggest that the enhancement of AA release in both U937 cells and macrophages may be caused by both increased cPLA2 activity and elevated cPLA2 protein expression. In addition, PMA stimulates PGE2 production via up-regulation of COX-1, and likely COX-2, expression in U937 cells whereas LPS stimulates PGE2 production via induction of COX-2 expression in macrophages.