Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. Fpassive was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Cardiomyocyte Fpassive was higher in HFPEF (7.6±0.4 kN/m2) than in AS (3.4±0.2 kN/m2; P<0.001) and in HFREF (5.1±0.2 kN/m2; P<0.001). In-vitro administration of PKG acutely lowered cardiomyocyte stiffness in all groups with the largest decrement in Fpassive in DHF patients. PKG activity in myocardial tissue homogenates was significantly lower in HFPEF (5.11±0.62 pmol/min/mg) than in both AS (9.18±0.64 pmol/min/mg; P<0.01) and HFREF (11.51±2.0 pmol/min/mg; P<0.001). Immunohistochemical determination of myocardial PKG activity by pVASP/VASP ratio provided confirmatory evidence as it was also significantly lower in HFPEF (0.70±0.03) than in both AS (0.84±0.02; P<0.001) and HFREF (0.85±0.03; P<0.001). Lower PKG activity in HFPEF than in aortic stenosis or HFREF was associated with higher cardiomyocyte Fpassive (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/ oxidative stress (P<0.05). Reduced PKG activity and lower myocardial cGMP concentration in HFPEF did not result from altered myocardial sGC or PDE5A expression, which was similar in all groups nor from unequal BNP expression, which was comparable in HFPEF and AS. The downregulated cGMP-PKG signaling in HFPEF was therefore related to low myocardial nitric oxide bioavailability because of high nitrosative/oxidative stress.