Creatine kinase enzymes are present in tissues such as muscle and brain to interconvert creatine phosphate and ADP, thus providing a system to interconnect energy production and utilization (Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862). Creatine kinase isoenzymes in kidney have received little attention since kidney contains relatively low creatine kinase activity compared with muscle and brain and because there is disagreement regarding the identity of the specific isoforms expressed in kidney. Using a combination of chromatographic and immunological techniques, we have identified two isoforms of creatine kinase in rat kidney supernatants, B creatine kinase, and the non-sarcomeric form of the mitochondrial creatine kinase, which represent 82 and 15%, respectively, of the total creatine kinase activity in this tissue. The identity of the non-muscle form of the mitochondrial creatine kinase was confirmed by N-terminal sequence analysis and compared with recently published cDNA sequences (Haas, R. C., and Strauss, A. W. (1990) J. Biol. Chem. 265, 6921-6927). We prepared multiple antisera specific for each isoform using synthetic peptide immunogens based upon nonhomologous regions from the primary sequence of each creatine kinase isoform. Immunocytochemical results demonstrate that both creatine kinase isoforms are colocalized in the inner stripe of the outer medulla in tubules of the distal nephron. A similar distribution of creatine kinase isoforms was obtained when different layers of the renal cortex and medulla were examined for creatine kinase activity and isozyme content using nondenaturing electrophoresis. In general, the distribution of creatine kinase enzymes in kidney corresponds to the regions of greatest ATP utilization, oxygen consumption, and sodium transport. These results suggest a role for creatine kinase enzymes in the coupling of ion transport and oxidative phosphorylation in the distal nephron of the mammalian kidney.