Poly(dA–dT)· poly(dA–dT) is a double‐helical B DNA containing A· T and T· A base pairs in alternating sequence. Although Raman spectra of this structure have been reported previously, the temperature dependence of the Raman bands has not been examined in detail. Using a spectrometer of high spectral precision and sensitivity, we applied Raman difference spectroscopy to determine the temperature dependence of all Raman bands of poly(dA–dT)· poly(dA–dT) in physiological salt solutions (both H₂O and D₂O) over the temperature range 10–85  °C. Three temperature domains are distinguished by Raman spectroscopy: premelting (10 < t < 66  °C), in which the double‐stranded structure is perturbed but does not dissociate; melting (66 < t < 75  °C), in which the double‐stranded structure dissociates; and postmelting (75 < t < 85  °C), in which no structural change can be detected. The results demonstrate that distinct Raman difference signatures exist for the premelting and melting transitions, and that each involves changes in most Raman bands. Among other novel findings and assignments: (i) Raman bands at 728 (dA), 1236 (dT) and 1301 cm⁻¹ (dA) provide the most sensitive measures of base stacking in both the premelting and melting domains. (ii) Raman bands at 1182 (dT) and 1512 cm⁻¹ (dA) provide the most sensitive measure of A· T unpairing in the melting domain. An unexpected finding is the apparent correlation of the 1182 cm⁻¹ band intensity with the degree of thymine unpairing. Additionally, bands at 1376 (dT) and 1577 cm⁻¹ (dA) confirm changes in interbase hydrogen bonding with premelting. (iii) The temperature dependence of the Raman band at 819 cm⁻¹ is closely correlated with those of backbone marker bands at 792 and 842 cm⁻¹ (O– P– O stretch modes), indicating that the 819 cm⁻¹ band is due to a vibrational mode localized in the backbone and is diagnostic of phosphodiester torsions α / ζ apparently specific to the gauche⁻/trans conformation of B_II DNA. (iv) The Raman band at 1484 cm⁻¹ (dA) is sensitive to hydrogen bonding at the adenine N1 site. (ν) Comparison of spectra from H₂O and D₂O solutions reveal significant vibrational coupling between Raman bands of the deoxynucleoside residues and B‐form backbone of poly(dA–dT)·poly(dA–dT). These results constitute an important foundation for future Raman studies of premelting and melting phenomena in other DNA sequences, including poly(dA)·poly(dT). Copyright © 1999 John Wiley & Sons, Ltd.