RSV infection of Hep-2 or HeLa cells leads to biochemical and morphological changes of cytoskeletal intermediate filaments (IF). Thus, human cytokeratin 18 is modified to generate a more acidic polypeptide of slightly larger apparent molecular weight. In addition, the amounts of vimentin and other cytokeratins are reduced, probably as a consequence of proteolytic degradation. These changes are reflected in a decrease of immunofluorescence with specific antibodies in RSV-induced syncytia and a more disorganized arrangement of IF arrays. About 50% of virus nucleoprotein (NP) is extracted with the high salt and detergent-insoluble intermediate filament fraction. Pulse-chase experiments indicate that NP needs a maturation period after synthesis to associate with IF. It is suggested that RSV needs to interact with IF during its life cycle and that association of NP, and/or other viral components, with IF might then lead to cytoskeletal structures becoming unstable in RSV-infected cells.