Toxic shock syndrome (TSS) is primarily caused by toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB). These toxins belong to a family of pyrogenic toxin superantigens (PTSAgs) produced by Staphylococcus aureus and exhibit several shared biological properties, including the induction of massive cytokine release and V-specific T-cell proliferation. The crystal structures of most PTSAgs are now published, and they demonstrate a striking similarity in conformational architecture even though their primary protein sequences are different. Despite these structural and immunobiological similarities, no cross-reactivity between TSST-1 and other PTSAgs has been demonstrated in serological or neutralization assays. Our laboratory has developed a neutralizing murine anti-TSST-1 monoclonal antibody (MAb5) which displayed cross-reactivity with SEB by enzyme-linked immunosorbent assay. The aim of the present study was to evaluate whether MAb5 can also cross-neutralize SEB-induced superantigenic activities in vitro. MAb5 was found to partially inhibit SEB-induced T-cell mitogenesis (63%) and tumor necrosis factor alpha (TNF-) secretion (70%) in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner, while an isotypic anti-TSST-1 monoclonal antibody showed no effect. Epitope mapping revealed that MAb5 bound to TSST-1 residues 47 to 56 (⁴⁷FPSPYYSPAF⁵⁶) and to SEB residues 83 to 92 (⁸³DVFGANYYYQ⁹²), sequences that located in different regions of these toxins and are structurally dissimilar. SEB peptide ⁸³DVFGANYYYQ⁹² was synthesized and found to also inhibit SEB-induced mitogenesis and TNF- secretion in human PBMC. Our results demonstrate for the first time that MAb5 binds to different epitopes on TSST-1 and SEB that appear functionally important in inducing T-cell mitogenesis and TNF- secretion in vitro.