13C-MRI HP 13C-bicarbonate (H13CO3-) and carbon dioxide (13CO2) is a novel technique for tissue pH mapping. Here, we report 13C-enriched ethyl acetyl carbonate (13C-EAC) for esterase-catalyzed production of HP-13CO2 and HP-H13CO3- for pH measurements. Our results showed that 13C-EAC was rapidly hydrolyzed by esterase to 13C-monoacetyl carbonate, which decomposed to HP-13CO2. Equilibrium between the newly produced 13CO2 and H13CO3- was established and the 13C-NMR signals can be quantified for pH measurements. Finally, in vivo pH measurements using H-13C-EAC was demonstrated in rat livers. These results suggest that HP-13C-EAC is a novel imaging probe for in vivo pH measurements of tissues.