The molecular processes underlying carcinogenesis and malignant spread are the foundation of future drug development for the treatment of cancer. Understanding these processes requires study of the interaction of complex biologic systems in a way that spatially and temporally recapitulates that seen in humans. Likewise, once an anticancer agent is developed, its intended antitumor action and its unintended side-effects must be studied in a rigorous and reproducible manner prior to its introduction into the clinic, a process that can benefit from methods that elucidate specific molecular processes and that can be performed serially. Recent advances in small-animal models of cancer, radiochemistry of single photon emitting radionuclides, single photon emission tomography systems, and image reconstruction techniques have set the stage for an ever-increasing use of SPECT and SPECT/CT in preclinical oncology-related applications. Several of these advances as well as several specific applications in oncology are highlighted and areas needing further improvement are identified.