Background: Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A₂ (cPLA₂) and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor (PPAR) agonist pioglitazone (PIO) limits IS, upregulates and activates cPLA₂ and COX2, and increases myocardial 6-keto-PGF₁ levels without activating eNOS and iNOS. We asked whether PIO also limits IS in eNOS^-/- and iNOS^-/- mice. Methods: Male C57BL/6 wild-type (WT), eNOS^-/- and iNOS^-/- mice received PIO 10 mg/kg/d (PIO+) or water alone (PIO-) for 3 days. Mice underwent 30min coronary artery occlusion and 4h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. Results: PIO reduced IS in the WT (15.4±1.4% vs. 39.0±1.1%; p<0.001), as well as in the eNOS^-/- (32.0±1.6% vs. 44.2±1.9%; p<0.001) and iNOS^-/- (18.0±1.2% vs. 45.5±2.3%; p<0.001) mice. The protective effect of PIO in eNOS^-/- mice was smaller than in the WT (p<0.001) and iNOS^-/- (p<0.001) mice. PIO increased myocardial Ser-633 and Ser-1177 P-eNOS levels in the WT and iNOS^-/- mice. iNOS was undetectable in all six groups. PIO increased cPLA₂, COX2 and PGI₂ synthase levels in the WT, as well as in the eNOS^-/- and iNOS^-/- mice. PIO increased myocardial 6-keto-PGF₁ levels, cPLA₂ and COX2 activity in the WT, eNOS^-/- and iNOS^-/- mice. Conclusions: The myocardial protective effect of PIO is iNOS independent and may be only partially dependent on eNOS. As eNOS activity decreases with age, diabetes and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.