Reproductive toxicity is one of the potential side effects of anticancer alkylating agents, with potential effects on vimentin intermediate filaments, one of the main components of the Sertoli cytoskeleton. Research suggests (Aumuller et al, 1988; Aumuller et al, 1992) that the highly organized and active Sertoli cytoskeleton is important in spermatogenesis. The aim of the current study was to investigate the effects of alkylating agents on vimentin filament expression in vitro. Sertoli cells, isolated from 20-day-old mice testes, were cultured for 5 days and then incubated with 0, 50, 100, and 200 micromol/L nitrogen mustard (HN2). Morphologic changes in Sertoli cells were observed per 30-minute interval at 12-hour exposure time points to 100 micromol/L HN2. Vimentin expression was investigated by immunocytochemistry at 6 hours and 24 hours posttreatment and reverse transcriptase polymerase chain reaction and Western blot at 12 hours posttreatment with 50, 100, and 200 micromol/L HN2. Exposure to HN2 resulted in a comparatively small Sertoli cell body with diminished cytoplasm. Sertoli cells were shrunk or detached. Cytoskeletal disruption increased with increasing HN2 concentration. The optical density values of vimentin antibody and expression of vimentin mRNA and protein were significantly decreased with increasing concentration of HN2. Significant treatment dose-dependent and time-dependent differences of vimentin mRNA and protein expression levels were also noted. Our data suggest that the change in the biochemical properties of vimentin may indicate that one of the mechanisms of reproductive toxicity resulting from HN2 is disruption of Sertoli cell vimentin filament structure, accompanied by a down-regulation of vimentin expression.