Myofilament basis mechanism of afterload-dependent increase in contractile force is still remained unclear (1). The purpose of this study is to investigate whether increasing afterload correlate with myosin head proximity to actin filaments and presumably crossbridge activated during contractions. We used eight male Wistar rats anesthetized by pentobarbital (50mg/kg, ip). We used x-ray diffraction analyses of beat-to beat contractions in spontaneously beating rat hearts under open-chest conditions simultaneous with recordings of left ventricular (LV) pressure and volume(2, 3). Aortic occluder was placed at descending aorta to increase afterload. We recorded x-ray diffraction patterns of the LV anterior free wall before and after aortic occlusion to determine the change in intensity ratio (I1,0/1,1) and myosin filament lattice spacing (D1,0). We found significant change neither in end-diastolic I1,0/1,1 nor D1,0, indicating that the proportion of myosin heads in proximity to actin filaments and myosin filament lattice spacing were unchanged by increasing afterload. Peak systolic I1,0/1,1 decreased significantly more after aortic occlusion than before aortic occlusion. Increase in D1,0 during contraction was significantly reduced after aortic occlusion than before aortic occlusion. A significant increase in myosin head proximity to actin filaments with increasing afterload correlated with increase in LV end-systolic pressure. A significant reduction in changes of D1,0 between diastole and systole with increasing afterload correlated with decrease in LV stroke volume. Since sarcomere length of cardiomyocyte related inversely with D1,0 because of constant cellular volume, reduced D1,0 change during contraction means prevention of sarcomere shortening with increasing afterload. These findings support a role of arterial resistance in determining LV stroke volume as the cardiovascular interaction.