¹³C-labeled pyruvate has been the most successful substrate to date for dynamic nuclear polarization (DNP) because of its essential role connecting multiple cellular metabolic pathways. A simple metric, ¹³C-lactate-to-¹³C-pyruvate ratio, is frequently used as it reflects lactate dehydrogenase (LDH) activity and intrinsic pool sizes. In addition, intracellular [lactate]:[pyruvate] is an important biomarker of cytosolic redox-state, directly reflecting free cytosolic [NADH]:[NAD⁺]¹. The ¹³C-lactate-to-pyruvate ratio derived from infused hyperpolarized ¹³C-pyruvate, however, does not accurately measure cellular redox-state since much of the labeled pyruvate and lactate is in extracellular space, and is often affected by vascular perfusion and substrate transport via monocarboxylate transporter (MCT). Both NADH and NADPH are key components in cellular anti-oxidation systems; NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation². While Keshari et al. and Bohndiek et al. estimated [NADPH]:[NADP⁺] using the balance of hyperpolarized ¹³C-dehydroascorbate and vitamic C^3,4, a more recent study showed large variations in cytosolic [NAD⁺]/[NADH] in different cancer cells using hyperpolarized ¹³C-glucose⁵. However, ¹³C-glucose suffers a significantly short T₁ even with deuteration and should go through multiple metabolic steps to produce pyruvate and lactate. Hyperpolarized [1-¹³C]alanine, which can be also transported across the plasma membrane⁶, is useful to measure intracellular pyruvate and lactate as the level of alanine transaminase (ALT) in the serum is low⁷. In this work, we propose a simple method to assess in vivo cytosolic redox-state using hyperpolarized [1-¹³C]alanine, and demonstrate the ethanol-induced redox change in rat liver.All measurements were performed on a clinical 3T GE Signa PET/MR scanner using GE SPINlab DNP polarizer. A custom-built transmit/receive ¹³C surface coil (Ø = 28 mm) was placed on top of the livers of healthy male Wistar rats (440-453g, N = 2). Each animal was anesthetized with 2-3 % isoflurane in oxygen (~1.5 L/min), then administered a solution of 80-mM hyperpolarized [1-¹³C]alanine for the measurement of baseline redox-state. To induce a hepatic redox change⁸, 1 mL/kg of 40-% ethanol was intravenously infused⁹, followed by the measurement of the perturbed redox-state using another hyperpolarized [1-¹³C]alanine. MRS data were acquired following the injection of the hyperpolarized compounds using the dynamic free induction decay sequence with a 10^o hard RF pulse (pulse width = 40 μs, spectral width = 5,000 Hz, 2,048 spectral points, acquisition time = 4 min, temporal resolution = 3 s). Independent phantom experiments were performed to estimate liquid polarization level and T₁ of ¹³C-labeled alanine.Hyperpolarized [1-¹³C]alanine samples were measured as ~20 % of liquid-state polarization and ~69 s of T₁, which is comparable to [1-¹³C]pyruvate T₁, after ~6 hrs of polarization. In vivo study showed lactate and pyruvate peaks in rat liver from an injection of hyperpolarized [1-¹³C]alanine (Fig. 1). Lactate-to-pyruvate ratio was measured from time-averaged (0-60s) spectra as 9.59 and 9.60 in two rats, which are consistent to the reported [lactate]/[pyruvate] in liver¹. Although reliable temporal change of lactate production could be measured, signal-to-noise ratio (SNR) of pyruvate was not sufficient enough to measure pyruvate kinetics, probably because of the small intrinsic pyruvate pool size. Figure 2 shows a time-averaged spectrum and time-courses of ¹³C-labeled metabolites acquired from a representative rat liver. One potential way to augment the pyruvate and lactate SNRs is to co-inject unlabeled pyruvate to increase the pyruvate pool size¹⁰. The ratio was increased to 26.00 and 17.31, respectively, 45-min after the ethanol infusion (Fig. 3). The amount of increase is also in the range of previously reported ethanol-induced redox change⁸.