The NMR coupling constants ((3)J(H-N, H-alpha)) of dipeptides indicate that the backbone conformational preferences vary strikingly among dipeptides. These preferences are similar to those of residues in small peptides, denatured proteins, and the coil regions of native proteins. Detailed characterization of the conformational preferences of dipeptides is therefore of fundamental importance for understanding protein structure and folding. Here, we studied the conformational preferences of 13 dipeptides using infrared and Raman spectroscopy. The main advantage of vibrational spectroscopy over NMR spectroscopy is in its much shorter time scale, which enables the determination of the conformational preferences of short-lived states. Accuracy of structure determination using vibrational spectroscopy depends critically on identification of the vibrational parameters that are sensitive to changes in conformation. We show that the frequencies of the amide I band and the A(12) ratio of the amide I components of dipeptides correlate with the (3)J(H-N, H alpha). These two infrared vibrational parameters are thus analogous to (3)J(H-N, H alpha), indicators for the preference for the dihedral angle phi. We also show that the intensities of the components of the amide III bands in infrared spectra and the intensities of the skeletal vibrations in Raman spectra are indicators of populations of the P-II, beta, and alpha(R) conformations. The results show that alanine dipeptide adopts predominantly a P-II conformation. the population of the beta conformation increases in valine dipeptides. The populations of the aR conformation are generally small. These data are in accord with the electrostatic screening model of conformational preferences.