To evaluate substrate utilization along the mouse nephron, cellular ATP content was measured under various conditions by the luciferin-luciferase technique. Individual micro-dissected nephron segments were incubated in a modified Hanks' solution (pH 7.4) with or without each of the following substrates: D-glucose, DL-lactate, beta-hydroxybutyrate (HBA), and L-glutamine. ATP production from glucose was minimal in the early proximal tubule (S1), but was substantial in the late proximal tubule (S3). Glutamine and lactate were the preferred substrates in proximal tubules. In contrast, ATP production from glutamine was less than that from the other substrates in distal nephron segments, including medullary and cortical thick ascending limbs of Henle's loop (MTAL and CTAL), distal tubule including the connecting tubule (DT), and cortical and medullary collecting tubules (CCT and MCT). Lactate and HBA were preferred substrates for ATP maintenance in CTAL, MTAL, and DT. Glucose was the best substrate in CCT. In addition, the specific contribution of anaerobic metabolism to maintaining cellular ATP was low in MTAL and CTAL. On the other hand, the glycolytic capacity of CCT and MCT was high. The above results demonstrate the substrate requirements for maintaining cellular ATP content within specific nephron segments.