The effect of rotavirus infection on cytoskeletal organization was examined in cultured African green monkey kidney (CV-1) cells. Rhesus rotavirus caused significant and selective changes in the organization of the vimentin filament network without having any effect on microtubules or actin. Double-immunofluorescence studies showed that at 6 h post-infection, and in the absence of cytopathic effect, the normal arrays of vimentin fibres radiating from multiple sites around the nucleus were lost. Vimentin fibres became irregularly distributed in the cytoplasm and were totally disrupted in the later stages of infection. Vimentin reorganization occurred independent of extracellular Ca2+ levels.