Translation of novel oncology positron emission tomography (PET) imaging tracers and tumour targeting agents into the clinic requires extensive preclinical studies in animal models. Our research combines traditional biodistribution analyses with state-of-the-art imaging technologies including small animal-PET imaging and high resolution autoradiography to provide information that may then be correlated with expression of specific biomarkers using methods such as immunohistochemistry (IHC). Small animal-PET imaging allows for whole body tomographic imaging at a resolution of approximately 2 mm. For higher resolution analyses of tracer uptake and biodistribution, high resolution digital autoradiography of whole body or tissue sections can then be performed using a Biospace Beta Imager (60 μm resolution), or Micro Imager (15 μm resolution), respectively. These imagers allow real-time autoradiography of β-emitting isotopes such as 3H, 14C and 32P as well as electrons associated with γ isotopes like 125I and positrons from PET tracers such as 18F. We have successfully employed these technologies for a range of applications. Using a combination of PET imaging and the Micro Imager, we evaluated the biodistribution, tumour uptake, retention and specificity of 18F-MEL050, a novel probe for melanoma imaging, in a B16 murine melanoma model. The favourable imaging results led to the rapid progression of the molecule into clinical trial. In another study, the biodistribution of novel Auger emitting DNA ligands conjugated to the somatostatin analogue, octreotate, was investigated using whole body autoradiography in mice bearing tumours over-expressing somatostatin receptor 2. The imaging data revealed robust uptake of the tracer into the tumour with minimal uptake into non-target organs. We have also utilised PET, autoradiography and IHC in a pilot study investigating intratumoral patterns of hypoxia in a mouse breast cancer model. Together, these studies demonstrate the application of these technologies in the preclinical development of molecular imaging and tumour targeting agents.