Following bolus injection of the superantigen Staphylococcus enterotoxin B (SEB) into mice, ligand-reactive T cells are triggered to release toxic amounts of lymphokines. Subsequently, within 6 to 10 h ligand-reactive Vbeta8+ T cells become anergic and clonally expand and thereafter are deleted via apoptosis. Since the binding affinities of SEB to major histocompatibility complex (MHC) class II molecules are as low as 1.7 microM, the concentration of SEB in the fluid phase dictates the presentation via MHC class II molecules. Here, we study the pharmacokinetics of SEB in vivo and correlate pharmacokinetics with immunogenicity. We describe here how after a bolus injection of SEB, the superantigen becomes systemically distributed, with peak levels within 5 to 30 min in blood and in lymph nodes. Clearance occurs within 10 to 24 h, with the kidneys being a major route. To induce T-cell activation in vivo, SEB must be present in concentrations above 10(-4) microg/ml. These concentrations exist for only 15 h. Manifestation of functional outcomes such as anergy, clonal expansion, and clonal deletion begins after 24 h. We conclude that the SEB model system can be used to separate the phase of T-cell receptor ligation from the phase of manifestation of functional outcomes.