A Raman linear intensity difference (RLID) method has been developed to determine orientations of chromophores in filamentous macromolecular assemblies. The method involves orientation of the filaments by hydrodynamic shear force and measurement of intensity differences between Raman spectra excited with laser polarizations parallel and perpendicular to the direction of orientation. Analysis of the RLID spectrum is simplified when the chromophore exhibits a vibrational mode for which the Raman band is enhanced by laser irradiation in resonance with a single molecular electronic transition. In the case of tryptophan, two such modes can be identified : The Raman bands corresponding to indole normal modes w2 and w16 gain intensity predominantly through resonance with L(a) and B-b electronic transitions, respectively, when excited by laser wavelengths of 266 and 240 nm. We have oriented the filamentous bacterial virus fd by hydrodynamic shear force in a velocity gradient flow cell and examined the orientation of the single indole ring (tryptophan-26) in the major coat protein subunit (pVIII) of the virion. The indole ring is found to be inclined at 31 +/- 4 degrees from the virion axis with its pseudo-2-fold axis at an angle of 38 +/- 6 degrees to the virion axis. Using the indole ring orientation determined by the RLID method and the side chain torsion chi(2,1) (dihedral angle C alpha-C beta-C3-C2) determined from the Raman w3 band, the conformation of the W26 side chain in the viral coat protein has been determined. Similar analysis has been applied to the tyrosine-21 and tyrosine-24 residues of the pVIII subunit. On the basis of the present results, a molecular model is proposed for the pVIII subunit of the native fd assembly. The model is also consistent with data from fiber X-ray diffraction analysis and polarized Raman microspectroscopy of oriented fd fibers.