The feasibility of blood-pool pinhole ECG gated SPECT was investigated in healthy mice to assess right and left ventricular function analysis. Anaesthetized (isoflurane 1-1.5%) adult CD1 mice (n=11) were analyzed after intravenous administration of 0.2 ml of 550 MBq of (99m)Tc human albumin. For blood-pool gated SPECT imaging, 48 ventral step and shoot projections with eight time bins per RR over 180 degrees with 64 x 64 word images were acquired with a small animal gamma camera equipped with a pinhole collimator of 12 cm in focal length and 1.5 mm in diameter. For appropriate segmentation of right and left ventricular volumes, a 4D Fourier analysis was performed after reconstruction and reorientation of blood-pool images with a voxel size of 0.55 x 0.55 x 0.55 mm(3). Average right and left ejection fractions were respectively 52+/-4.7% and 65+/-5.2%. Right end diastolic and end systolic volumes were significantly higher compared with the corresponding left ventricular volumes (P<0.0001 each). A linear correlation between right and left stroke volumes (r=0.9, P<0.0001) was obtained and right and left cardiac outputs were not significantly different 14.2+/-1.9 and 14.1+/-2 ml/min, respectively.