The structure of the packaged double-stranded DNA genome of bacteriophage T7 was compared to that of unpackaged T7 DNA using digital difference Raman spectroscopy. Spectral data were obtained at 25 degrees C from native T7 virus (100 mg/mL), empty T7 capsids (50 mg/mL), and purified T7 DNA (40 mg/mL) in buffer containing 200 mM NaCl, 10 mM MgCl2, and 10 mM Tris at pH 7.5. At these conditions, the local conformation of T7 DNA was not affected by packaging. Specifically, the local B-form secondary structure of unpackaged T7 DNA, including furanose C2'-endo pucker, anti glycosyl torsion, Watson-Crick base pairing, and base stacking, were essentially fully (> 98%) retained when the genome was condensed within the viral capsid. However, the average electrostatic environment of T7 DNA phosphates was altered dramatically by packaging as revealed by large perturbations in the Raman bands associated with localized vibrations of the DNA phosphate groups. The change in the phosphate environment was attributed to Mg2+ ions that were packaged with the genomic DNA, and the observed Raman perturbations of genomic DNA were equivalent to those generated by a 50-100-fold increase in Mg2+ concentration in aqueous phosphodiester model compounds. The T7 data were qualitatively and quantitatively similar to those observed previously for packaged DNA of bacteriophage P22 and imply that genomic DNAs of T7 and P22 are both organized in a similar fashion within their respective capsids. The results show that the condensed genome does not contain kinks or folds that would disrupt the local B conformation by more than 2%. The present findings are discussed in relation to previously proposed models for condensation and organization of double-stranded and single-stranded viral DNA.