Nutrient demand is a fundamental characteristic of rapidly proliferating cells. Vitamin B12 is vital for cell proliferation; thus neoplastic cells have an increased demand for this essential nutrient. In this study we exploited the vitamin B12 uptake pathway to probe the nutritional demand of proliferating cells with a radiolabeled B12 derivative in various preclinical tumor models. We describe the synthesis and biological evaluations of copper-64-labeled B12-ethylenediamine-benzyl-1,4,7- triazacyclononane-N,N′,N′′-triacetic acid (B 12-en-Bn-NOTA-64Cu), the first example of a B12 derivative for positron emission tomography (PET) imaging. Small-animal imaging and pharmacological evaluation show high tumor uptake ranging from 2.20 to 4.84-%-ID-g-1 at 6 h post-administration. Competition studies with excess native B12 resulted in a 95-% decrease in tumor accumulation, indicating the specificity of this radiopharmaceutical for B12 endocytotic transport proteins. These results show that a vitamin B12 PET radiopharmaceutical has potential utility for non-invasive imaging of enhanced nutrient demand in proliferating cells. Co-opting cobalamin: Vitamin B12 is an essential nutrient for all proliferating cells. Rapid proliferation is a common hallmark of cancer; consequently, neoplastic cells have an increased demand for vitamin B12. We exploited the vitamin B12 uptake pathway to non-invasively probe the nutrient demand of cancer cells. The synthesis, characterization, and tumor-homing properties of a vitamin B12 PET radiotracer are described. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.