The detection of aminopeptidase N (APN) activity can give information on tumor development. Hyperpolarized L-[1-13C]alaninamide is a specific, sensitive probe of APN activity in kidney homogenate. Here, we characterized its in vivo metabolic response in the rat kidney. In addition to being an APN substrate, L-[1-13C]alaninamide is sensitive to pH and also reacts with dissolved carbon dioxide. To avoid spectral overlap, alaninamide is best suited as an APN probe in acidic environments, and it may have additional applications as a multifunctional sensor of pH and CO2.