Glycolysis, hypoxia, and proliferation are important factors in the tumor microenvironment contributing to treatment-resistant aggressiveness. Imaging these factors using combined functional positron emission tomography and computed tomography can potentially guide diagnosis and management of cancer patients. A dog with fibrosarcoma was imaged using 18F-FDG, 64Cu-ATSM, and 18F-FLT before, during, and after 10 fractions of 4.5 Gy radiotherapy. Uptake of all tracers decreased during treatment. Fluctuations in 18F-FDG and 18F-FLT PET uptakes and a heterogeneous spatial distribution of the three tracers were seen. Tracer distributions partially overlapped. It appears that each tracer provides distinct information about tumor heterogeneity and treatment response.