This study is intended to evaluate the feasibility of using a high-resolution pinhole SPECT system and iodine-123-N-isopropyl-4-iodoamphetamine ((123)I-IMP) for three-dimensional (3D) absolute quantitation of regional cerebral blood flow (rCBF) in mice. The pinhole SPECT system consists of a rotating stage and a pinhole collimator attached to a clinical gamma camera. The collimator's focal length is 251 mm. Phantom studies were performed to evaluate sensitivity and full-width half-maximum (FWHM) spatial resolution. The aperture-to-object distance was 15 mm. Six mice were studied. Cerebral infarctions were induced by ligating and disconnecting the distal portion of the left middle cerebral artery. Ex vivo SPECT studies were performed using harvested brains and skulls. The CBF volumetric image was computed using the standardized input function. Excellent spatial resolution of 0.9-mm FWHM and uniform sensitivity throughout the 3D volume were demonstrated in the phantom experiments. The CBF images showed a defect in the infarcted areas and a reduction of CBF values in the infarcted region as compared with the control region. This study demonstrated the feasibility of the 3D quantitation of rCBF in mice using a high-resolution pinhole SPECT system and (123)I-IMP.