A novel equilibrium hydrogen exchange Fourier transform IR (HX-FTIR) spectroscopy method for predicting secondary structure content was employed using spectra obtained for a training set of 23 globular proteins. The IR bandshape and frequency changes resulting from controlled levels of H-D exchange were observed to be protein-dependent. Their analysis revealed these variations to be partly correlated to secondary structure. For each protein, a set of 6 spectra was measured with a systematic variation of the solvent H-D ratio and was subjected to factor analysis. The most significant component spectra for each protein, representing independent aspects of the spectral response to deuteration, were each subjected to a second factor analysis over the entire training set. Restricted multiple regression (RMR) analysis using the loadings of the principal components from 19 of these H-D analyses revealed an improvement in prediction accuracy compared with conventional bandshape-based analyses of FTIR data. Nearly a factor of 2 reduction in error for prediction of helix fractions was found using s 1 , the average spectral response for the H-D set. In some cases, significant error reduction for prediction of minor components was found using higher factors. Using the same analytical methods, prediction errors with this new deuteration-response-FTIR method were shown to be even better than those obtained by use of electronic circular dichroism (ECD) data for helix predictions and to be significantly lower for ECD-based sheet prediction, making these the best secondary structure predictions obtained with the RMR method. Tests of a limited variable selection scheme showed further improvements, consistent with previous results of this approach using ECD data.