A specialized kinetic analysis of real-time hyperpolarized [1,1,2,2-D₄, 1-¹³C]choline ¹³C-magnetic resonance spectroscopy enabled the determination of initial rates of metabolic enzyme activity (choline oxidase), enzyme–substrate affinity (K_m), and inhibition. In a clinical MRI scanner, metabolite levels lower than 16 μM were detected at a temporal resolution of 1 s.