We report and interpret the first Raman spectrum of a double-stranded RNA virus containing a membrane envelope. Spectra of the native bacteriophage θ6 and of its isolated host-attachment (spike) protein and phospholipid-free core assembly were collected from aqueous solutions over a wide range of temperature. Comparison of the vibrational spectra by digital difference methods permits the following structural conclusions regarding molecular constituents of the fully assembled virion. (1) The double-stranded RNA, phospholipid and protein components of the phage exhibit Raman amplitudes in accordance with their biochemically determined compositions in the native virion (10, 20 and 70%, respectively). (2) α-Helix and irregular conformations are the dominant secondary structures in proteins of both the viral membrane and nucleocapsid. This represents a departure from previously examined icosahedral phage and plant viruses, which are dominated by β-sheet structures. (3) The phospholipids of the viral membrane are liquid crystalline throughout the determined range of virus thermostability (0 to 40°C). (4) The P3 spike protein of θ6, which is anchored to, but not sequestered within the viral membrane, is largely α-helical (∼35%) and highly thermolabile. Denaturation of P3 at temperatures above 30°C leads to appreciable loss (∼ 20%) of α-helix in favor of β-strand structure, and alters significantly the environments of many aromatic side-chains. (5) The secondary structures of integral membrane proteins of θ6 are overwhelmingly α-helical (∼70 to 80%) and also thermolabile. In contrast to P3, which exhibits aspartate and glutamate carboxyls in the ionized form (CO-2), the integral membrane proteins exhibit only protonated carboxyl groups (COOH).Treatment of θ6 with butylated hydroxytoluene (BHT), which has been shown to remove the P3 spike protein, does not significantly perturb phospholipids and associated integral proteins of the viral membrane or structural proteins and packaged double-stranded RNA of the nucleocapsid. However, P3 subunits, which are recovered after BHT treatment, exhibit radically altered secondary and tertiary structures, including the loss of most subunit α-helices. Among the P3 side-chains affected by BHT treatment, we note a general trend toward greater hydrophilicity and greater solvent exposure of the aromatic residues Trp and Tyr. On the other hand, the cysteine sulfhydryl groups of the BHT-isolated P3 monomer are not solvent exposed and function as strong hydrogen-bond donors in the protein core. The BHT-mediated structure transition of P3 is discussed in relation to putative quaternary rearrangements involving the membrane anchor protein, P6, and homomultimer complexes of the P3 monomer.The present studies show that RNA, nucleocapsid and viral membrane components of an enveloped double-stranded RNA virus can be distinguished and structurally characterized by Raman spectroscopy. Importantly, we have demonstrated the ability to obtain Raman spectra from the intact viral membrane, establishing the feasibility of Raman spectroscopy for structural analysis of protein molecules embedded in a viral phospholipid bilayer. The results obtained also serve as the basis for detailed spectroscopic analyses of the θ6 nucleocapsid, the θ6 polymerase complex (nucleocapsid core), and the packaged θ6 genome, as described in the accompanying paper (Bamford et al., 1993).