The staphylococcal enterotoxins (SE) specifically bind to class II major histocompatibility complex (MHC) proteins, resulting in activation of monocytes and T cells. The SE cause weight loss in mice, which is dependent on T-cell stimulation and tumor necrosis factor alpha (TNF-α) production. Here we use a mutant of staphylococcal enterotoxin A that binds class II MHC molecules and activates monocytes but not T cells to evaluate the relative contributions of monocyte- and T-cell-stimulatory activities to in vivo toxicity. The mutant toxin did not cause weight loss in B10.BR mice but did stimulate monocyte TNF-α production in vitro, as did the wild-type toxin. Addition of a supernatant from toxin-activated T cells enhanced monocyte- stimulatory activity of both mutant and wild-type toxins fivefold. The effect of the supernatant could be mimicked by recombinant gamma interferon (IFN-γ) and was inhibited by antibody to IFN-γ. These results suggest that toxin- induced monocyte TNF-α production is upregulated by IFN-γ, which likely represents the T-cell requirement in SE-mediated weight loss. Our studies thus implicate two distinct class II MHC-dependent signaling pathways for SE, the first involving direct signal transduction through class II MHC molecules mediated by either mutant or wild-type toxin and the second requiring T-cell stimulation by toxin-class II MHC complexes with consequent production of IFN-γ. We suggest that both pathways are required for optimal monocyte TNF- α production in vitro and SE-induced toxicity in vivo.