Mitochondrial adenine nucleotide translocation was measured in the presence of creatine or creatine phosphate to test the proposed functional couple between the nucleotide translocase and creatine kinase. Rat heart mitochondria were preloaded with the corresponding nucleotide which reacts with creatine kinase only after it is transported across the mitochondrial membrane; namely, radioactive ATP-preloaded mitochondria were assayed in the presence of 10 mM creatine plus ADP, and radioactive ADP-preloaded mitochondria in the presence of 10 mM creatine phosphate plus ATP. Results showed that forward creatine kinase reaction (in the direction of creatine phosphate synthesis) inhibits translocation of external ADP into mitochondrial matrix and backward reaction (cleavage of creatine phosphate to creatine) likewise inhibits translocation of ATP across the mitochondrial membrane. Control experiments showed that without the kinase activity when Mg2+ was omitted from the assay medium, the presence of creatine or creatine phosphate had no effect on ADP or ATP transport, respectively. Therefore, the observed inhibition of nucleotide exchange by these compounds is due to creatine kinase activity which upon reacting with the newly exported nucleotide can effectively compete for transport back into the mitochondrial matrix over nucleotides added in the assay medium. Kinetic analysis also indicated that the forward creatine kinase reaction inhibits external ADP uptake competitively. These results are interpreted to support the proposal that a functional interaction exists between the mitochondrial bound creatine kinase and the adenine nucleotide translocase.