The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5,4, resolution. The icosahedral CVB3 panicles crystallize in the monoclinic space group, P21, (a--574.6, b = 302.1, c---521.6A, /3--107.7 °) with two virions in the asymmetric unit giving 120-fold non-crystallographic redundancy. The crystals diffracted to 2.7 A resolution and the X-ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self-rotation function established pseudo R32 symmetry with each particle sitting on a 32 special position. This constrained the orientation and position of each panicle in the monoclinic cell to near face-centered positions and allowed for a total of six possible monoclinic spacegroup settings. Correct interpretation of the highresolution (3.0-3.2~,) self-rotation function was instrumental in determining the deviations from R32 orientations of the virus particles in the unit cell. Accurate particle orientations permitted the correct assignment of the crystal space-group setting amongst the six ambiguous possibilities and for the correct determination of particle positions. Real-space electron-density averaging and phase refinement, using human rhinovius 14 (HRV14) as an initial phasing model, have been carried out to 3.5,~ resolution. The initial structural model has been built and refined to 3.5 ~, resolution using X-PLOR.