Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and ¹⁸F-labeled 1-(2′-deoxy-2′-fluoroarabinofuranosyl) cytosine] ([¹⁸F]FAC), a PET probe recently developed by our group. We show that [¹⁸F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [¹⁸F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [¹⁸F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [¹⁸F]FAC microPET scans can predict responses to dFdC. We suggest that [¹⁸F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.