In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same "quality" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging "voxel" as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-[2-[4-(2-methoxylphenyl) piperazino]ethyl]-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6-1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of approximately 5-50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the "true" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice.