This review is focused on metabolic mapping in biological tissue with quantitative bioluminescence and single photon imaging. Metabolites, such as ATP, glucose and lactate, can be imaged quantitatively and within microscopic dimensions in cryosections from shock frozen biological specimens using enzyme reactions and light emission by luciferases. The technique has been applied in numerous targets and models of experimental biomedical research, such as multicellular spheroids, various organs of laboratory animals in a physiological or pathophysiological state, and even in plant seeds. Among numerous other aspects, data obtained with this method have contributed to the elucidation of mechanisms that are involved in the development of necrosis in multicellular spheroids. The combination of the bioluminescence technique with immunohistochemistry, autoradiography or in situ hybridization can considerably reduce ambiguities in the interpretation of the experimental results. Although, an invasive technique, bioluminescence imaging has been used most intensively in clinical oncology using tumor biopsies taken at the first diagnosis of the disease. It has been shown for squamous cell carcinomas of the head and neck and of the uterine cervix that accumulation of high levels of lactate in the primary lesions is associated with a high risk of metastasis formation and a reduced overall and disease-free patient survival. Thus, metabolic imaging can provide additional information on the degree of malignancy and the prognosis of tumors which may help the oncologist in improving specific treatment approaches for each individual malignant disease. Last but not least, metabolic mapping in clinical oncology has stimulated a number of investigations in basic cancer research on mechanisms that underlie the correlation between tumor metabolism and malignancy.