Researchers long have relied on research involving small animals to unravel scientific mysteries in the biological sciences, and to develop new diagnostic and therapeutic techniques in the medical and health sciences. Within the past 2 decades, new techniques have been developed to manipulate the genome of the mouse, allowing the development of transgenic and knockout models of mammalian and human disease, development, and physiology. Traditionally, much biological research involving small animals has relied on the use of invasive methods such as organ harvesting, tissue sampling, and autoradiography during which the animal was sacrificed to perform a single measurement. More recently, imaging techniques have been developed that assess anatomy and physiology in the intact animal, in a way that allows the investigator to follow the progression of disease, or to monitor the response to therapeutic interventions. Imaging techniques that use penetrating radiation at millimeter or submillimeter levels to image small animals include x-ray computed tomography (microCT), single-photon emission computed tomography (microSPECT), and imaging positron emission computed tomography (microPET). MicroCT generates cross-sectional slices which reveal the structure of the object with spatial resolution in the range of 50 to 100 microns. MicroSPECT and microPET are radionuclide imaging techniques in which a radiopharmaceutical is injected into the animal that is accumulated to metabolism, blood flow, bone remodeling, tumor growth, or other biological processes. Both microSPECT and microPET offer spatial resolutions in the range of 1-2 millimeters. However, microPET records annihilation photons produced by a positron-emitting radiopharmaceutical using electronic coincidence, and has a sensitivity approximately two orders of magnitude better than microSPECT, while microSPECT is compatible with gamma-ray emitting radiopharmaceuticals that are less expensive and more readily available than those used with microPET. High-resolution dual-modality imaging systems now are being developed that combine microPET or microSPECT with microCT in a way that facilitates more direct correlation of anatomy and physiology in the same animal. Small animal imaging allows researchers to perform experiments that are not possible with conventional invasive techniques, and thereby are becoming increasingly important tools for discovery of fundamental biological information, and development of new diagnostic and therapeutic techniques in the biomedical sciences.