Background: In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide-(specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)-stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling. Methods and Results: Mice with cardiac myocyte inducible PDE5 overexpression (P5) were crossed to those lacking nitric oxide synthase 3 (N3), and each model, the double cross, and controls were subjected to transaortic constriction. P5 mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3 mice were protected. However, P5/N3 mice behaved similarly to P5 mice despite the lack of nitric oxide synthase 3-coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide-stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5 hearts exhibited higher oxidative stress, whereas P5/N3 hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling. Conclusions: These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide-derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease. [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-].