The pro-inflammatory mediator cyclooxygenase-2 and its product PGE₂ are induced in the ischemic heart, contributing to inflammatory cell infiltration, fibroblast proliferation and cardiac hypertrophy. PGE₂ synthesis coupled to COX-2 involves two membrane-localized prostaglandin E synthases, mPGES-1 and mPGES-2; however, it is not clear how these synthases are regulated in cardiac myocytes and fibroblasts. To study this, we used primary cultures of neonatal ventricular myocytes (VM) and fibroblasts (VF) treated with interleukin-1 (IL-1) for 24 hrs. To test for involvement of MAPKs in IL-1 regulation of mPGES-1 and-2, cells were pretreated with pharmacological inhibitors of p42/44 MAPK, p38 MAPK and c-jun kinase (JNK). mRNA was analyzed by RT-PCR. Protein was analyzed by densitometry of Western blots. mPGES-1 was undetectable in untreated VF, but induced by IL-1, and inhibition of either p42/44 MAPK or JNK, but not p38 MAPK, was almost completely inhibitory. In VM, inhibition of the 3 MAPKs reduced IL-1-stimulated mPGES-1 protein by 70 - 90%. mPGES-2 was constitutively synthesized in both VM and VF and was not regulated by IL-1 or MAPKs. Confocal microscopy revealed co-localization of both mPGES-1 and mPGES-2 with COX-2 in the perinuclear area of both VF and VM. Finally, PGE₂ production was higher in VM than VF. Our data show that: 1) mPGES-1 is induced in both VF and VM; 2) regulation of mPGES-1 by MAPK family members is different in the two cell types; 3) mPGES-2 is constitutively synthesized in both VM and VF and is not regulated; and 4) mPGES-1 and -2 are co-localized with COX-2 in both cells. Thus differences in activity of mPGES-1 and COX-2 or coupling of COX-2 with mPGES-1 may contribute to differences in PGE₂ production by myocytes and fibroblasts.