High-resolution, non-invasive, 3D-imaging techniques would greatly benefit the investigation of the localization properties of tumor-specific radiopharmaceuticals in laboratory animals. The present study reports how pinhole SPECT can be applied to tumor localization studies in small laboratory animals to provide high resolution SPECT images in vivo. Pinhole SPECT was performed using a rotating scintillation camera, equipped with a pinhole collimator. The sensitivity of a 2 mm diameter collimator at 45 mm from the source is 90 cps/MBq for 99mTc. The planar spatial resolution at a 45 mm distance is 2.2 mm. The transaxial spatial resolution, with a distance of 45 mm between the collimator aperture and the axis of rotation, is 3.1 mm. For SPECT imaging, spatial linearity is preserved across the usable field-of-view. The major advantage of the high resolution properties of pinhole tomography is demonstrated by the enhanced lesion-to-normal-brain uptake ratio achieved on tomographic slices as compared to planar images. For example, 201Tl tumor-to-normal-brain uptake ratios of 1.1 to 1.3 observed on planar images, corresponded to ratios ranging from 3.2 to 3.7 on the SPECT slices. Examples of the activity distributions of two radiopharmaceuticals in tumor and in normal brain for sagittal and coronal images are given. In all cases, tumors are clearly delineated on the pinhole SPECT slices. The present study shows that pinhole SPECT performed with standard SPECT instrumentation can give high spatial resolution images, with a FWHM approximately 3 mm and a sensitivity approximately 100 cps/MBq for 99mTc.