Multiple model systems have demonstrated that negatively selected thymocytes can be deleted during the immature CD4(+)CD8(+) CD3(low) stage after high affinity interaction of T-cell receptors (TCRs) with antigen:major histocompatibility complex (MHC) complexes. Superantigens (SAGs) derived from endogenous mammary tumour viruses (Mtv) induce negative selection of Mtv-SAG-reactive thymocytes regardless of which peptide antigen is presented by MHC molecules. In this study, the timing of deletion of multiple subsets of Mtv-SAG-reactive CD4(+)CD8(+) thymocytes was investigated by a 4 colour flow cytometry in SJL x CBA/J cross-bred mice. Deletion of V beta 3(+), V beta 5(+), V beta 11(+), and V beta 17(+) Mtv-SAG-reactive thymocytes was found to occur synchronously in the most mature CD3(medium) and early CD3(high) subsets of CD4(+)CD8(+) thymocytes, in contrast with reports showing that the deletion of Mtv-SAG-reactive thymocytes can occur at different stages in particular model systems.