Raman spectra of calf thymus DNA were measured in the pH interval 6.4 to 3.45 in the presence of divalent manganese ions. pH-dependent protonation of AT and GC base pairs and conformational changes were indicated in the spectra. Protonation of adenine residues becomes obvious at pH 4.4 and continues upon lowering the pH to 3.45. Adenine protonation is connected with the disruption of AT base pairs. Protonation of GC base pairs is indicated at somewhat lower pH than that of AT base pairs, namely at pH 3.8, and continues upon lowering the pH to 3.45. At pH 3.8 unstacking of thymine residues is indicated, and spectral markers for the unstacking of adenine and cytosine were found at pH 3.45. Changes of the DNA backbone are indicated by spectral changes of conformational marker bands at 898 and 1423 cm−1. Copyright © 2005 John Wiley & Sons, Ltd.