Renal gluconeogenesis contributes to glucose homeostasis and is elevated in diabetes. Aspartate is an efficient gluconeogenic substrate in the kidney, and its conversion to glucose proceeds via phosphoenolpyruvate carboxykinase (PEP-CK), which is a rate-limiting enzyme. Scanning the kidney of rats infused with hyperpolarized [1-13C]aspartate, the metabolites detected include [1-13C]malate and [4-13C]malate, 3-phospho[1-13C]glycerate, and a trace of bicarbonate. Using [1,4-13C2]aspartate resulted in higher bicarbonate signal, consistent with PEP-CK activity, and bicarbonate was undetectable after inhibiting PEP-CK. Compared to fed rats, the bicarbonate-to-malate ratio was 3-fold higher in fasted rats, indicating the potential of hyperpolarized aspartate to probe renal gluconeogenesis.