Kidney primarily oxidizes fatty acids and ketone bodies for ATP production but the substrate preference is altered in metabolic diseases. Renal metabolism is generally evaluated in kidney slices and in vivo. However, real-time metabolism has not been studied in isolated perfused kidneys where substrate availability and pharmacological interventions can be well-controlled. Here, we investigate pyruvate metabolism in isolated perfused rat kidneys by hyperpolarized 13C NMR. Results show that hyperpolarized 13C-pyruvate was rapidly metabolized in the kidneys and productions of 13C-bicarbonate, 13C-alanine, and 13C-lactate were detected. the utilization of pyruvate for energy production was confirmed by isotopomer analyses of tissue extracts.