Using a large variety of readily available and investigative radiopharmaceuticals and relatively inexpensive instrumentation, single-photon emission computed tomography (SPECT) imaging provides a useful tool to obtain valuable functional information in vivo. Limited by the use of lead collimation, clinical SPECT has much lower detection efficiency and poorer spatial resolution resulting in general inferior image quality as compared to positron emission tomography (PET). However, in imaging small organs or small animal, pinhole collimation can be used for improved spatial resolution and a substantial increase in detection efficiency. With the use of lower-energy single-photon isotopes, SPECT is not limited by positron range and other instrumentation factors that limit the spatial resolution of PET. With appropriately designed pinhole collimator and aperture, calibration method and image reconstruction techniques, micro-SPECT systems can be designed to achieve spatial resolution on the order of 1 mm. Further advance in multi-detector pinhole SPECT systems will allow practical high-resolution micro-SPECT imaging of small animals.