NMR relaxation dispersion spectroscopy is a powerful technique to elucidate the mechanism of protein-protein binding reactions. However, it is difficult to optimize the concentration ratios that give relaxation dispersions of appropriate amplitude to determine accurate kinetic and thermodynamic parameters, especially in cases of very tight binding. In this study, we have obtained N-15 R (2) dispersions of Asn803-hydroxylated hypoxia-inducible factor-1 alpha (HIF-OH) in the presence of a substoichiometric amount of its target protein, the transcriptional adapter zinc-binding (TAZ1) domain of CREB binding protein, whereas no R 2 dispersion was observed for the bound state of HIF-OH at 1:1 concentration ratio because the binding is too tight. Although the R (2) dispersions were measured for the free peaks of HIF-OH, they enabled us to quantitate the kinetic and thermodynamic parameters of HIF-OH/TAZ1 binding process. Simulations of effective R (2) rates revealed that the association rate is the key factor to determine the amplitude of R (2) dispersions. By careful optimization of the concentration ratio, the R (2) dispersion method should be generally applicable for studying a wide range of protein-protein, protein-nucleic acid, and protein-small molecule interactions.