Two-dimensional1H exchange spectroscopy has been applied to the studies of imidazole binding to cytochromec.The magnetization exchange rate constants, the substitution reaction rate constants and the equilibrium constants have been evaluated. Some resonances of imidazole substituted forms (Im-cytc) have been assigned by 2D exchange NMR. The observed hyperfine shifts of heme methyl groups of Im-cytcand cytchave been corrected for contact and dipolar contributions. The detailed analysis of these results indicates that the axial ligands and their orientations, and the substituents of heme are important determinants of the electronic structure of the heme in cytcand its derivatives.