AbstractN,N‐Bis(2‐fluorophenyl)carbamoyl chloride inactivates the serine protease α‐chymotrypsin by stoichiometric carbamoylation, presumably at the active site serine‐195 residue. At sample temperatures above 25°C fluorine NMR spectra of this enzyme derivative reveal the presence of conformations which are interchanged by processes that have ΔG 25≠of about 70 kJ mol−1as estimated by two independent methods. Fluorine NMR spectra of the protein dissolved in 8 M urea were characterized by shift and relaxation parameters which suggest an unfolded state for the protein but with interconversion kinetics similar to those of the native structure. At sample temperatures below 25°C a second kinetic process becomes apparent in the spectra of both the native and denatured protein, although it was not possible to quantitate the rate of this process. These spectral changes are discussed in terms of the likely conformational properties of theN,N‐bis(2‐fluorophenyl) group attached to the native enzyme and are shown to be consistent with one of the 2‐fluorophenyl rings having a high barrier to rotation, and thus rotating slowly, while the other rotates rapidly. Both rings rotate rapidly in the denatured protein. A form of the modified enzyme which had fluorine NMR characteristics similar to those of the denatured enzyme yet was fairly soluble in 0.05 M sodium chloride solution was detected in samples after extended heating; formation of this species was not readily reversible. Spin‐lattice relaxation times (T1) and19F{1H} NOE data for the native and denatured modi