Nivalenol (NIV), a trichothecene mycotoxin, is a secondary fungal metabolite mainly produced by Fusarium nivale. We first reported that NIV could induce apoptosis and changes in lymphocyte subsets in lymphoid tissues of mice. In this study, to clarify the direct effects of NIV on thymocytes, mouse thymocyte primary cultures were treated with NIV at the dose levels of 0.25, 0.5 and 1.0 μg/ml and examined for up to 24 h after treatment by flow cytometry. The number of viable cells decreased significantly at and after 6 h in dose- and time-dependent manners, and FACS analysis revealed that the apoptotic cell index showed a significant increase in all treated groups at and after 3 h in a time-dependent manner. The index at 24 h was lowest in 1.0 μg/ml-group. The number of CD4 + CD8 + cells was prominently depleted in all groups in a time-dependent manner. On the other hand, the numbers of CD4 + CD8 - and CD4 - CD8 + cells were significantly depleted only in 1.0 μg/ml-group at 24 HAT. These results indicate that NIV directly affects thymocytes and induces apoptosis mainly in CD4 + CD8 + cells.