Methods were devised or modified which made it possible to measure phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase in seven defined parts of single nephrons and in patches from thin limb and papilla areas dissected from freeze-dried microtome sections of rat kidney. All three enzymes were essentially confined to the proximal tubule. In normal kidneys, the levels were highest in the proximal convoluted tubule. Glucose-6-phosphatase was 20 times higher in the early part of the convoluted segment than in the late part of the straight segment. With one exception, in acidosis, only phosphoenolpyruvate carboxykinase increased (fourfold in the proximal convoluted segment but much less in the straight portion). In starvation, phosphoenolpyruvate carboxykinase increased about as much as in acidosis in the proximal straight tubule, but not as much in convoluted portions, whereas glucose-6-phosphatase rose modestly in both parts of the proximal tubule and fructose bisphosphatase rose only in the straight tubule, especially the early segment. It is suggested that ammoniagenesis can accompany gluconeogenesis in the proximal convoluted tubule but not in the straight segment.