Radiochemical microprocedures were developed for the determination of hexokinase and phosphoenolpyruvate carboxykinase (PEPCK) activity in single microdissected segments of the mature rabbit nephron dissected from fresh tissue after collagenase treatment. All results were related to tubular length and tubular protein content. Hexokinase activity was found to be lowest in the proximal convoluted tubule and to increase along the following nephron segments, with highest activity in the connecting tubule. The gluconeogenic enzyme PEPCK, on the other hand, was exclusively found in the proximal tubule. Early and late portions of the convoluted segment exhibited the same specific activity, but only 50% was found in the pars recta. All other renal structures exhibited only insignificant activity of PEPCK. The results show that renal glucose metabolism and gluconeogenesis are clearly separated. As previously shown for the cytosolic rat enzyme, rabbit mitochondrial PEPCK is also exclusively a proximal tubular enzyme, thus confirming the dominant role of this segment in mammalian renal gluconeogenesis. The high activity of hexokinase in the segments of the distal tubule points to the role of glucose as metabolic fuel, glycogen precursor, and other glucose-6-phosphate-using pathways in these structures.