The effects of vomitoxin (VT) on the binding activity of three transcription factors critical to pro-inflammatory cytokine regulation were assessed in the RAW 264.7 murine macrophage model by electrophoretic mobility shift assay (EMSA). When cells were treated with 100 to 250 ng/ml of VT, activator protein-1 (AP-1 binding) was increased after 2 and 8 h. This effect was potentiated when cells were coincubated with lipopolysaccharide (LPS) (synchronous model) but not when preincubated with LPS (delayed synchronous model). Supershift EMSA revealed that VT preferentially induced JunB, JunD, phosphorylated c-Jun, c-Fos, and Fra-2 binding activities of the AP-1 family. Nuclear factor kappaB (NF-kappaB) binding was increased at 2 and 8 h in cells subjected to synchronous and delayed synchronous VT exposure in the presence of LPS. Supershift EMSA indicated that the p-50 and c-Rel subunits of NF-kappaB/ Rel were specifically affected. Nuclear factor-IL6 (NF-IL6) binding was increased at 2 and 8 h with or without LPS in synchronous and delayed synchronous VT-exposure models. Here, the C/EBPbeta subunit was primarily involved in enhanced NF-IL6 binding. The capacity of VT to elevate binding of AP-1, NF-kappaB, and NF-IL6 may contribute to the VT-mediated cytokine up-regulation in vitro and in vivo. The observations that VT was active in synchronous and delayed synchronous models suggest that macrophages activated simultaneously or prior to toxin exposure were vulnerable to the effects of this trichothecene.