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Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-¹³C]pyruvate that the lactate dehydrogenase–catalyzed flux of ¹³C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using ¹³C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.