In this report we examined overlap between superantigen (SAg) and Toll-like receptor 4 (TLR4) stimulation of the innate immune system. Before in vivo stimulation we found that mouse splenic DCs expressed unexpectedly low levels of surface TLR4 compared to macrophages. In response to LPS, TLR4 gene expression in fractionated spleen cells was downregulated. By comparison, surface TLR4 staining with the Sa15-21 mAb showed little downregulation, and the anti-TLR4 MTS510 mAb showed decreased staining, suggesting that LPS was bound to TLR4 at the time points examined. Interestingly, SAg stimulation induced decreased TLR4 staining as measured by the MTS510 mAb, even though the TLR4 gene was not downregulated. Nevertheless, LPS potently induced DCs to produce TNF and IL-12, but SAg did not, even though they efficiently activated DCs. Notwithstanding, in vivo stimulation with staphylococcal enterotoxin SAg conditioned the innate immune system to hyper-respond to various pathogen-associated molecular patterns (PAMPs). Specifically, pre-priming with SAg enhanced LPS-mediated DC synthesis of TNF and IL-12. Thus, SAgs may exert their pathogenesis on the host by conditioning DCs, in a T cell activation dependent manner to potentiate responses to PAMPs.