In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K(+)-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K(+)-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in non-phagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.