Measurements of the nuclear Overhauser effect (NOE), which is a nuclear magnetic resonance (NMR) double resonance technique, for the H-2 proton on ADP have been used to identify the amino acid residue binding ADP at the active site of creatine kinase. Application of a strong radio-frequency field H2 at a frequency of 0.9 ppm or 1.7 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate results in a negative NOE for the H2-2 proton resonance of ADP in its complex with creatine kinase. The magnitude of the NOE for the ADPH-2 proton depends on the ratio of ADP to creatine kinase binding site concentration; the dependence indicates that there is rapid exchange between free and bound ADP. Comparable values of the NOE for the H-2 proton of adenosine 5'-mono-, 5'-di-, and 5'-triphosphate and inosine 5'-diphosphate in binary complexes with creatine kinase show that the binding site for these nucleotides is the same. The large negative NOE for the H-2 proton of ADP is maintained for the various binary, ternary, quaternary, and pentenary complexes of creatine kinase with ADP formed by addition of the activator Mg(II), the other substrate creatine, and the planar anion nitrate which is an inhibitor. These results indicate that the conformational changes known to occur upon addition of the other ligands do not involve the entire active site. In particular, the environment around the nucleotide is unperturbed. Inactivation of creatine kinase by reaction with iodoacetamide causes considerable conformational changes. However, as indicated by the large negative NOE for the H-2 proton of ADP in a binary complex with the inactivated enzyme, the environment around the base is altered minimally. Experiments were performed to identify the proton groups on the enzyme, resonating at 0.9 and 1.7 ppm, which interact with the ADPH-2 proton. An NOE was not observed when the aromatic protons of the enzyme were irradiated with the strong radiofrequency field H2 implying that aromatic protons are not near the H-2 proton of ADP in the enzyme complex. The H-2 proton of 1-N6-ethenoadenosine 5'-diphosphate, an analogue of ADP with the 1-nitrogen and 6-nitrogen blocked from potentially hydrogen bonding, still exhibits a large NOE in the nucleotide-enzyme complex. The indication is that the protons promoting the H-2 proton NOE are not on an amino acid residue which binds ADP by hydrogen binding. Creatine kinase was inactivated by reacting the single essential arginyl residue per subunit with diacetyl. No NOE for the H-2 proton of ADP in the presence of the inactivated enzyme was observed. Observation of the H-2 proton resonance of the inhibitor adenosine in the presence of the enzyme revealed no NOE in contrast to the observations with the adenine nucleotides.