Two enzymes of ketone body metabolism, 3-oxoacid CoA-transferase and 3-hydroxybutyrate dehydrogenase were measured with ultramicromethods to map their activities along single, dissected segments of mouse nephron. 3-Oxoacid CoA-transferase activity was assayed with a radiochemical procedure by separating [14C]succinyl-CoA, formed in the presence of acetoacetyl-CoA from [1,4-14C]succinate. This procedure, when compared to the spectrophotometric method, resulted in similar activities in mouse organ homogenates. 3-Hydroxybutyrate dehydrogenase activity was determined with sufficient sensitivity by using NADH-dependent luciferase. The dehydrogenase exhibited only 5% of the activity of the 3-oxoacid CoA-transferase when measured in mouse kidney cortex homogenates. Both enzymes were found to be present in all nephron structures studies. The specific activity related to tubular protein, which was determined in parallel in all segments dissected, exhibited a typical distribution pattern along the nephron. Both enzymes roughly paralleled each other along the structures of the distal nephron. They exhibited high activities in the thick ascending limb of Henle's loop and the distal convoluted tubule, but decreased to nearly 20% in the segments of the collecting tubule. In the proximal convoluted and straight tubule 3-oxoacid CoA-transferase activity was almost equal. In contrast, 3-hydroxybutyrate dehydrogenase increased by a factor of 5 from the convoluted to the straight portion. This heterogeneity along the proximal tubule remained when pure D-hydroxybutyrate was used as substrate, but was not found when rat or rabbit nephron segments were analysed. Lowest activities of enzymes were recovered from glomeruli and thin descending limbs of Henle's loop. The results obtained allow for the conclusion that 3-hydroxybutyrate and acetoacetate can be metabolized in all structures of the mouse nephron with different capacities in various segments. With the exception of the relatively low activity of 3-hydroxybutyrate dehydrogenase in the proximal convoluted tubule, the distribution pattern mirrors the distribution of mitochondria along the rat nephron. The results point to the possible role of ketone bodies as energy fuels for nephron segments performing active transport processes.