A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi 29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi 29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi 29 DNA packaging motor.