Molecular imaging of small animals in vivo is vital in the study of mouse and rat models of human diseases, and will provide important clues to the pathogenesis, progression and treatment of many disorders. Functional imaging of small animals using ultra-high resolution single photon emission tomography (SPECT) should be a valuable tool in the molecular imaging armamentarium. SPECT has been used to study cerebral binding sites, to image the expression of reporter genes, and in applications in cardiology and oncology. In this review, we summarize the most recent developments in SPECT imaging of small animals, with particular reference to the types of systems available, their application, and some of the potential limitations.