Hyperpolarized molecular probes can be effectively used as pH markers. To date, the only two probes reported in vivo as extracellular pH sensors are 13C-bicarbonate, and [1,5-13C2]zymonic acid. Alaninamide is a derivative of alanine which is found to be sensitive to variations of pH in the physiological range. The aim of the present study was to assess the feasibility of using alaninamide as a pH probe in vivo. The alaninamide titration curve was determined by performing 13C NMR measurements at 9.4 T, 37° on a set of 500 mM Ala-NH2∙HCl samples of varying pH referenced to 13C urea. [1-13C]Alaninamide was polarized at 1 K in a 7 T polarizer, then rapidly dissolved in a buffered solution and injected IV into a Sprague Dawley rat (n=6) located in a 9.4 T animal scanner. 13C FIDs were acquired with 30° BIR4 pulses using a single loop 1H / quadrature 13C surface coil placed over the left kidney. The pH was perturbed by injecting acetazolamide IV (10 mg/kg) one hour prior to infusion. The alaninamide titration curve shows a 13C1 chemical shift change of ≈ 8.4 ppm, and a pKa of 7.9. The pH sensitivity of 13C1 results in three distinct alaninamide spectral peaks, corresponding to three different extracellular pH compartments within the kidney (pH = 7.46, pH = 7.22, pH = 6.58) that can be tentatively assigned to the cortex/blood, medulla and calyx/ureter. With acetazolamide treatment, the pH in the first compartment follows the change in pH of the blood, while the pH in the third compartment does not reflect the urine pH and shifts during the brief experiment. No change is observed in the pH value of the second compartment.