The binding of γ-picoline to horse heart ferricytochromec(cytc) by displacing methionine-80 has been studied using two dimensional exchange spectroscopy (EXSY) method. Some hyperfine shifted resonances arising from heme peripheral protons in the γ-picoline complex of cytc(γ-MePy·cytc) have been assigned. The rate and equilibrium constants of γ-picoline binding to cytchave been measured and the thermodynamic values of ΔH°, ΔS° and the activation energy Eafor the forward reaction have been obtained. The patterns of the hyperfine shifts of γ-MePy·cytchave been illustrated and the reason for the low affinity of γ-picoline to cytchas been discussed.