Glomerular synthesis of prostaglandin E₂ (PGE₂) and the identification of factors that control its production are important for understanding diseases such as membranous nephropathy, nephrotic syndrome and anti-Thy1 nephritis. We investigated the signaling pathways that regulate both the synthesis and actions of PGE₂ in glomerular podocytes. To study the actions of PGE₂, we assessed the ability of this eicosanoid to regulate phorbol ester (PMA)-stimulated [³H]arachidonic acid (AA) release from a mouse podocyte cell line. PMA dose-dependently increased cPLA₂-mediated AA release over 60 minutes, while PGE₂ dose-dependently reduced PMA-stimulated AA release to a maximum of 33%. We investigated the E-Prostanoid (EP) receptor-signaling pathway required for such inhibition. PGE₂ dose-dependently increased cAMP levels in an EP₂ receptor-dependent manner, while the cAMP-elevating agents, forskolin (FSK) and isobutylmethylxanthine (IBMX) reproduced the inhibitory actions of PGE₂ on PMA-stimulated AA release. RpcAMPs, a protein kinase A (PKA) inhibitor, reversed these effects. We next evaluated PGE₂ synthesis in this podocyte cell line. After 60 minutes, PMA increased PGE₂ levels 4-fold, and upregulated COX-2 expression while leaving COX-1 levels unchanged. However, this acute PGE₂ synthesis was significantly reduced by the COX-1-selective inhibitor, SC-560, and to a lesser extent by the COX-2-selective inhibitor, SC-58125. Our findings suggest that PMA-stimulated PGE₂ synthesis in mouse podocytes requires both basal COX-1 activity as well as induced COX-2 expression, and that PGE₂ reduces PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such an autocrine regulatory loop might have important consequences for podocyte and glomerular function in the context of renal diseases involving PGE₂ synthesis.