BACKGROUND High resolution spatial details of the distribution of activity in three dimensions is required to evaluate the localization and dosimetric properties of radiolabelled monoclonal antibodies in tumors and normal tissues. Planar imaging of small animals with a resolution of 5-10 mm is usually the imaging modality of choice. The authors investigated high resolution single-photon emission computed tomographic (SPECT) imaging, based on a rotating pinhole scintillation camera. Although the sensitivity of the pinhole collimator is low, several radionuclides offer suitable decay properties to perform pinhole SPECT, especially in conjunction with high activity levels used in radioimmunotherapy. METHODS Transverse, sagittal, and coronal sections were reconstructed using a three-dimensional cone-beam algorithm, which is a generalization of the two-dimensional fan-beam filtered backprojection algorithm. Before reconstruction, the pinhole projections were corrected for the decay of the radionuclide, geometric and intrinsic efficiency variations of the camera system, and center of rotation shift. RESULTS The spatial resolution at 50 mm from the pinhole collimator with 3.3 mm aperture was 3.4 mm, and the sensitivity 7.2 c/s microCi for technetium-99m. With the 2 mm collimator the resolution was 2.2 mm, and the sensitivity was 2.6 c/s/microCi. To show the spatial resolution in vivo, a rat was injected with 185 MBq of technetium-99m-methylene diphosphonate or with 5 mCi technetium-99m-hexamethylpropylene amine oxime. The bone structures were well delineated in the methylene diphosphonate image, and in the hexamethylpropylene amine oxime image, the brain was nicely shown. For comparison a magnetic resonance image for the same section was done. CONCLUSIONS High resolution SPECT imaging with the pinhole collimator provides mapping of the activity in three-dimensions, needed for more detailed biodistribution data and to perform more accurate dosimetry.