Activation of the innate immune system might predispose a host to toxicant-induced inflammation. In vitro macrophage models were employed to investigate the effects of preexposure to Toll-like receptor (TLR) agonists on induction of proinflammatory cytokine gene expression by the trichothecene mycotoxin deoxynivalenol (DON) and other toxicants. Priming of the murine RAW 264.7 macrophage line or peritoneal murine macrophages with the TLR4 agonist lipopolysaccharide (LPS) at 100 ng/ml for 4, 8, and 16 h significantly increased DON-induced IL-1β, IL-6, and TNF-α mRNA expression as compared to LPS or DON alone. The minimum LPS concentration for sensitization of both cell types was 1 ng/ml. LPS priming also potentiated IL-1β mRNA induction by DON in human whole-blood cultures, suggesting the relevance of the murine findings. As observed for LPS, preexposure to TLR agonists including zymosan (TLR2), poly (I:C) (TLR3), flagellin (TLR5), R848 (TLR7/8), and ODN1826 (TLR9) sensitized RAW 267.4 cells to DON-induced proinflammatory gene expression. Amplified proinflammatory mRNA expression was similarly demonstrated in LPS-sensitized RAW 264.7 cells exposed to the microbial toxins satratoxin G, Shiga toxin, and zearalenone as well as the anthropogenic toxicants nickel chloride, triphenyltin, 2,4-dinitrochlorobenzene, and 2,3,7,8-tetrachlorodibenzodioxin. The results suggest that prior TLR activation might render macrophages highly sensitive to subsequent induction of proinflammatory gene expression by xenobiotics with diverse mechanisms of action.