Rationale: Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO-sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload-induced hypertrophy. The latter itself depresses signaling through this pathway by reducing NO generation and enhancing cGMP hydrolysis. Objective: We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms. Methods and Results: C57BL/6 mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, whereas NO-and heme-independent sGC activation by BAY 60-2770 was preserved. Total sGCα 1 and β 1 expression were unchanged by TAC; however, sGCβ 1 subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2-to 3-fold greater in Cav3-containing lipid raft versus nonlipid raft domains in control and 6-fold greater after TAC. In contrast, BAY 60-2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO-and BAY-stimulated sGC activity in microdomains containing Cav3 for controls but no change within non-Cav3-enriched domains. Conclusions: Pressure overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.