Significant effort is currently being devoted to the development of noninvasive imaging systems that allow in vivo assessment of biological and biomolecular interactions in mice and other small animals. Ideally, one would like to discern these functional and metabolic relationships with in vivo radionuclide imaging at spatial resolutions approaching those that can be obtained using the anatomical imaging techniques (i.e., <100 µm), which would help to answer outstanding questions in many areas of biomedicine.In this paper, we report progress on our effort to develop high-resolution focusing X-ray and gamma-ray optics for small-animal radionuclide imaging. The use of reflective optics, in contrast to methods that rely on absorptive collimation like single-or multiple-pinhole cameras, decouples spatial resolution from sensitivity (efficiency). Our feasibility studies have refined and applied ray-tracing routines to design focusing optics for small animal studies. We also have adopted a replication technique to manufacture the X-ray mirrors, and which in experimental studies have demonstrated a spatial resolution of ∼190 µm. We conclude that focusing optics can be designed and fabricated for gamma-ray energies, and with spatial resolutions, and field of view suitable for in vivo biological studies. While the efficiency of a single optic is limited, fabrication methods now are being developed that may make it possible to develop imaging systems with multiple optics that could collect image data over study times that would be practical for performing radionuclide studies of small animals.