Lactate production was measured directly in individual segments of the rat nephron. Tubules were dissected and then incubated in vitro with glucose as the only metabolic substrate. Each segment was incubated with and without antimycin A, an inhibitor of oxidative metabolism. Proximal tubules produced no lactate with or without antimycin A. The distal segments all produced lactate. The rate of lactate production without antimycin A ranged from 0.4 to 0.9 pmol X min-1 X mm-1 in all distal segments except one, the inner medullary collecting duct, which produced lactate at the significantly higher rate of 2.8 pmol X min-1 X mm-1. Antimycin A increased lactate production significantly in all of the distal segments. The increase was largest in medullary thick ascending limbs (1,400%) and cortical (798%) and outer medullary collecting ducts (357%). Increments were smaller in cortical thick ascending limbs (98%) and distal convoluted tubules (98%) and least in the inner medullary collecting ducts (28%). We conclude that lactate production occurs only in distal segments of the nephron and that under anoxic conditions significant amounts of ATP are produced by anaerobic glycolysis in these segments.