The present study aims at investigating the intrathymic maturational stage of T cells at which clonal deletion can be induced. Staphylococcal enterotoxin B (SEB) was added to organ cultures of murine fetal thymus lobes at various time points of culture, and V beta 8-expressing cells were assayed on day 14. V beta 8 low-expressing (V beta 8lo) cells were reduced to 40-60% of the control receiving no SEB, though the reduction was ambiguous when SEB was given on day 13. In marked contrast, V beta 8 high-expressing (V beta 8hi) cells were virtually completely deleted in all groups including the group given SEB on day 13. Most of the V beta 8hi cells that were deleted by 24 h of treatment with SEB were shown to be of the CD4+8- mature phenotype, though CD4-8+V beta 8hi cells were also deleted. It was further shown that the thymic V beta 8hi CD4+8- cells recovered from organs cultured for 14 days without SEB responded to immobilized anti-V beta 8 monoclonal antibody, indicating that V beta 8hi cells, which were highly sensitive to clonal deletion, were functionally competent mature T cells. These results strongly suggest that the thymus is capable of eliminating all T cells recognizing antigen present in the thymus regardless of the maturational stage of T cells.