The determination of protein secondary structure in aqueous solution has undergone an important advance [ 11. It is now possible to estimate fractions of bi-H-bonded andmonoH-bonded helix, antiparallel P-sheet, parallel P-sheet, and P-turn to within 25% of X-ray diffraction estimates with -70% confidence, whereas before, using circular dichroism (CD) [ 21 or Raman spectroscopy [3], only fractions of total helix and P-sheet were estimated, to within -8%. This improvement has come about mostly through the generation of computer-subtracted difference laser Raman spectra, and specifically through the subtraction of the water spectrum from the amide I spectrum of proteins. Here we present evidence that this general approach can be applied to the amide III’ region of the laser Raman spectrum of proteins in D20 as well, and that estimates of the structure of solvent exchanged regions of the protein can thereby be obtained. When a protein is dissolved in D,O, exposed amide N-H groups exchange with the solvent, and the .amide III vibrations shift to a lower frequency from between 1200 and 1320 cm-’ to what are termed the amide III’ vibrations between 900 and 1000 cm-‘. While several investigations have pursued analyzing the amide III region of the vibrational spectrum for protein secondary structure determination [3-71 little has been done with the amide III’ region. We believe that there are some practical advantages to using the amide III’ over the amide III region for structural studies: (i) Bands due to helix in the amide III region are not very intense relative to the corresponding nonhelical bands (R. W. W., A. K. D., W. L. P., in preparation). (ii) CH deformation modes are strong in the amide III helix region. The amide III’ region seems to be less affected by these kinds of problems. Both helical and S-sheet polypeptides show strong amide III’ bands in DzO. The C-C stretching bands in this region are also conformationally sensitive, and may constructively reinforce the structurally corresponding amide III’ bands. Here, the spectrum of each protein in DzO was subtracted from the corresponding spectrum of the protein in Hz0 so that bands unaffected by deuteration of the amide nitrogen are removed. The resulting difference spectrum represents the true contours of the amide III’ spectrum. Four of these amide III’ difference spectra [6] were then used to calculate the spectra of helix and P-sheet by a procedure similar that in [2] for CD spectra. We have performed these same calculations on the watersubtracted amide I spectra of these proteins in HzO, a method developed more fully in [ 1 ] so that a direct comparison can be made between the results of these two methods.