The vibrational Stark effect measures the effect of an external electric field on the vibrational (IR) spectrum of a molecule. This technique gives quantitative information on the sensitivity of a vibrational peak position to an electric field. This calibration can be used to evaluate shifts in the vibrational frequency caused by changes in the local electric field in the organized electrostatic matrix of a protein, for example, by mutating amino acid residues near the vibration whose frequency is probed. We report vibrational Stark effect measurements for NO bound to several distal pocket mutants of myoglobin, (Va168Asp, Va168Asn, Va168Glu, and His64Val). These mutations were designed to perturb the electrostatic field near the NO bound to the heme iron. The magnitude of the change in dipole moment, /Delta mu/, for the vibration of hTO bound to heme is found to be approximately 0.12 D/f, that is, the Stark tuning rate is 2.0/fcm(-1)(MV/cm) (where f is the local field correction) for a series of distal pocket mutants for which the vibrational frequency, <()over bar>(NO), varies by over 60 cm(-1) and also for a picket fence model compound. Both /Delta mu/ and the dispersion of <()over bar>(NO) are similar to those reported for CO bound to the heme iron (Park, E. S.; Andrews, S. S.; Hu, R. B.; Boxer, S. G., J. Phys. Chem. B 1999, 103, 9813-9817), This correlation can be quantitatively explained if the dispersion in <()over bar>(NO) and <()over bar>(NO) is modeled as an electrochromic band shift due to the interaction of the change in dipole moment of the oscillator and the electric field of the protein. The slope of the correlation is given by the measured ratio Delta mu (NO)/Delta mu (CO) obtained from the vibrational Stark effect data.