The dramatic advances of biological research in recent years that have focused on the molecular basis of how systems of the body (e.g. cells, organs and the whole organism) function, have increased the need for molecular imaging instrumentation. Of the several imaging modalities available today applied for in vivo studies of research animals, positron emission tomography (PET) is a technique that permits non-invasive use of positron labeled molecular imaging probes to image and assay biochemical processes of cellular function in the living subject. Imaging can be performed repeatedly before and after interventions and therefore allows the use of each animal as its own control. Many different positron labeled compounds have been and continue to be synthesized as probes that target a range of molecular targets within specific biochemical pathways. These molecular imaging probes are used in extremely low mass amounts, such that biological processes involving compounds in nanomolar concentration or lower can be imaged without disturbing the process. Biological processes from receptors and synthesis of transmitters in cell communication pathways, to metabolic processes and gene expression can be imaged. In the past, PET in animal research has been used extensively for studies of primates and larger animals. In recent years, the development of new detector technology has lowered the limits of spatial resolution. This has made it possible to use PET scanning for the study of the most important modern molecular biology model, the laboratory mouse. This paper presents some of the challenges facing small animal PET technology, provides an overview of the development of small animal PET systems, and discusses the current state of the art technology, some of its applications, as well as some future directions.