We introduced trifluoromethyl (CF) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using F NMR spectroscopy. On the basis of the anti-Curie behavior of CF signals, we found that the deoxy Mb is in thermal equilibrium between the B, (d)(d)(d)(d)(d), and E, (d)(d)(d)(d)(d), states of the heme Fe(II), i.e., B ⇆ E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼-20 kJ mol and ∼-60 J K mol, respectively, for the thermal equilibrium. Thus, the E state is slightly dominant over theB one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.