1H NMR studies were performed on the three redox states of Megasphaera elsdenii flavodoxin. The results show that the protein is remarkably stable, as concluded from amide proton exchange studies. Some amide protons are still present in the 1H NMR spectrum even after one month in 2H2O at 33 degrees C (pH 8.3). The reactivity of the exchangeable protons can be grouped into three categories, i.e. t1/2 much greater than 5 min, 10 s approximately less than t1/2 approximately less than 5 min, and t1/2 much less than 10 s. The amide proton exchange reactions are hardly dependent on the redox state. Optimal resolution of 1H NMR spectra is obtained at 33 degrees C, independent of the redox state. No conformational change of the protein is observed in the pH range between 6 and 8.5. Assignments of resonances to protons of flavin and of some amino acid residues are established in both the oxidized and the hydroquinone state using chemically and isotopically substituted flavins and the driven nuclear Overhauser technique. Preliminary two-dimensional 1H-1H correlated spectra show that the protein is amenable to two-dimensional NMR techniques. Previous assignments are confirmed by this technique.