Ex vivo and in vivo studies on liver metabolism using hyperpolarized [1-13C]pyruvate report do not agree on whether hyperpolarized bicarbonate metabolite production results from pyruvate oxidation or gluconeogenesis. This study tested the ability of hyperpolarized [1-13C]pyruvate to probe gluconeogenesis in the liver of intact rats. While conversion to hyperpolarized bicarbonate was detected in the liver of fasted rats, treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinc acid resulted in 7-fold lower levels. This result supports the notion that hepatic gluconeogenic metabolism can indeed be directly probed in vivo with hyperpolarized pyruvate.