In order to investigate how β-stimulation affects the contractility of cardiac muscle, x-ray diffraction from cardiac muscle in the left ventricular free wall of a mouse heart was recorded in vivo. This is the first x-ray diffraction study on a heart in a living body. After the R-wave in electrocardiograms, the ratio of the intensities of the equatorial (1,0) and (1,1) reflections decreased for about 50 msec from a diastolic value of 2.1 to a minimum of 0.8, and then recovered. The spacing of the (1,0) lattice planes increased for about 90 msec from a diastolic value of 37.2 nm to a maximum of 39.1 nm, and then returned to the diastolic level, corresponding to about 10% stretch of sarcomere. Stimulation of β-adrenergic receptor by dobutamine (20 μg/kg/min) accelerated both the decrease in the intensity ratio, which reached a smaller systolic value, and the increase in the lattice spacing. However, the intensity ratio and spacing at the end-diastole were unchanged. The recovery of the lattice spacing during relaxation was also accelerated. The mass transfer to the thin filaments at systole in a β-stimulated heart was close to the peak value in twitch of frog skeletal muscle at 4 °C, showing that the majority of cross-bridges have been recruited with few in reserve. [J Physiol Sci. 2006;56 Suppl:S57]