Prostaglandin E2 (PGE2) secretion during Leishmania infection has been reported. However, the signalling mechanisms mediating this response are not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to various stimuli, the implication of these enzymes was evaluated in Leishmania-infected phorbol myristate acetate-differentiated U937 human monocytic cell line. Time-course experiments showed that PGE2 synthesis increased significantly in parallel with COX-2 expression when cells were incubated in the presence of Leishmania donovani promastigotes or lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only detected when cells were stimulated with LPS. Indomethacin, genistein, and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, inhibited PGE2 production induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA synthesis, and there was a significant correlation between PGE2 inhibition and reduced COX-2 expression. Collectively, our results indicate that infection of U937 by L. donovani leads to the generation of PGE2 in part through a PKC-dependent signalling pathway involving COX-2 expression. They further reveal that PTK-dependent events are necessary for Leishmania-induced PGE2 generation, but not for COX-2 expression. A better understanding of the mechanisms by which Leishmania can induce PGE2 production could provide insight into the pathophysiology of leishmaniasis and may help to improve therapeutic approaches.