Objectives. Hyperemic myocardial blood flow (MBF) is impaired in hypertrophic cardiomyopathy (HCM) despite normal epicardial coronary arteries, which indicates microvascular dysfunction. The aim of the present study was to determine the relative contribution of extravascular compressive forces to microvascular dysfunction in HCM. Methods. Eighteen patients with symptomatic HCM and normal coronary arteries and 10 age matched healthy volunteers were studied with PET to quantify resting and hyperemic MBF at a subendocardial and subepicardial level. In HCM patients, MRI was performed to determine LV mass (LVMI) and volumes, echocardiography to assess diastolic perfusion time, heart catheterization to measure outflow tract gradient (LVOTG) and LV pressures, and serum NT-proBNP as a biochemical marker of LV wall stress. Results. Hyperemic MBF was blunted in HCM vs. controls (2.26 ± 0.97 vs. 2.93 ± 0.64 mL / min / g, p<0.05). In contrast to controls (1.38 ± 0.15 to 1.25 ± 0.19, p=NS), the endo-to-epi MBF ratio decreased significantly in HCM during hyperemia (1.20 ± 0.11 to 0.88 ± 0.18, p<0.01). This pattern was similar for hypertrophied septum and lateral wall. Hyperemic MBF was inversely correlated with LVOTG, NT-proBNP, left atrial volume index, and LVMI (all p<0.01). Multivariate regression analysis, however, revealed that only LVMI and NT-proBNP were independently related to hyperemic MBF, with greater impact at the subendocardial myocardial layer. Conclusions. Hyperemic MBF is more severely impaired at the subendocardial level in HCM patients. The level of impairment is related to markers of increased hemodynamic LV loading conditions and LV mass. These observations suggest that, next to reduced capillary density caused by hypertrophy, extravascular compressive forces contribute to microvascular dysfunction in HCM patients.