The recent availability of pre-clinical PET/SPECT/CT tri-modality systems provides an opportunity to image animals injected with mixed modality (PET/SPECT) isotopes. However, before these procedures can be truly useful, some understanding of the effect of different modality radiotracers on image quality and the resulting noise characteristics must be investigated. Our goal in this study was to characterize effects on image resolution that might arise in one modality because of the presence of another (different modality) isotope. A phantom, consisting of a set of side-by-side microcapillary tubes, were imaged on a trimodality (SPECT/PET/CT) pre-clinical scanner. Each tube was filled with a SPECT isotope and imaged while varying acquisition time, amount of activity, and distance between line sources. Images were evaluated to determine the visualization threshold at which objects became apparent in cross-sectional and MIP images (both that the objects were line sources and that the lines were two separate objects). These experiments were repeated in the presence of a PET isotope. After re-evaluating resolution, the signal to noise characteristics of the image as a whole were assessed and related to amounts of each isotope as well as total activity in the field of view. PET acquisition in the presence of SPECT isotopes was investigated using a contrast phantom with spheres filled with PET, SPECT, and mixed isotopes in backgrounds of mixed SPECT and PET isotopes. The noise properties of the background and spheres were measured as well as the visibility of the spheres. Finally, SPECT, PET, and CT images were acquired of a mouse after injection of both a PET and SPECT isotope. Acquisition of SPECT and PET images in the presence of different modality tracers (with careful considerations of the activity and acquisition parameters) seems feasible.