Right handed nucleotide helices exist in two main conformational families, A and B, which differ structurally at the molecular level. We are using UV resonance Raman spectroscopy to observe the B_A transition, induced by the addition of TFE to reduce relative hydration. Comparison of 240 nm excited UVRR spectra with spectra excited at 260 nm indicate A-T base pairs adopt the A-form structure at a lower relative hydration than G-C base pairs (74% vs 85% RH). UVRR spectra obtained as a function of decreasing water activity reveal a frequency shift of the dG ribosyl mode, indicative of a change in sugar pucker from C₂?-endo to C₃?-endo, consistent with a conversion from the B to A form. These data indicate that the conversion from C²?-endo to C³?-endo sugar pucker occurs at a lower hydration level (62% RH), relative to other structural changes, and may be the final step in the transition. Results obtained from MD simulation indicate that sugar pucker, base pair inclination, and X-axis displacement convert from A to B form values at discrete times (250 ps, 500 ps, and 1500 ps respectively), with conversion from A to B DNA complete by ∼1500 ps. Site specific base pair information is obtained using fluorescence spectroscopy, to further examine how location and base pair type contribute to the A to B transition. Solution and MD studies suggest a sequential mechanism for the B-A transition in DNA, and indicate the need for a re-evaluation of the current models for the transition.