Sildenafil, a selective inhibitor of phosphodiesterase-5, induces powerful protection against myocardial ischemia-reperfusion injury through activation of cGMP-dependent protein kinase (PKG). We further hypothesized that PKG-dependent activation of survival kinase, ERK, may play a causative role in sildenafil-induced cardioprotection via induction of eNOS/iNOS and Bcl-2. Our results show that acute intra-coronary infusion of sildenafil in Langendorff isolated mouse hearts prior to global ischemia-reperfusion significantly reduced myocardial infarct size (from 29.4±2.4% to 15.9±3.0%; P<0.05). Co-treatment with ERK inhibitor, PD98059, abrogated sildenafil-induced protection (31.8±4.4%). To further evaluate the role of ERK in delayed cardioprotection, mice were treated with sildenafil (i.p.) 24 hours before global ischemia-reperfusion. PD98059 was administered (i.p.) 30 min prior to sildenafil treatment. Infarct size was reduced from 27.6±3.3% in controls to 7.1±1.5% in sildenafil-treated mice (P<0.05). The delayed protective effect of sildenafil was also abolished by PD98059 (22.5±2.3%). Western blots revealed that sildenafil significantly increased phosphorylation of ERK1/2 and GSK3, and induced iNOS, eNOS, Bcl-2 and PKG in the heart 24 hours after treatment. PD98059 inhibited the enhanced expression of iNOS, eNOS, and Bcl-2 and phosphorylation of GSK3. PD98059 had no effect on sildenafil-induced activation of PKG. We conclude that these studies provide first direct evidence that PKG-dependent ERK phosphorylation is indispensable for the induction of eNOS/iNOS and Bcl-2 and the resulting cardioprotection by sildenafil.