While non-stimulated primary human monocytes exhibit very low levels of tumor necrosis factor (TNF)-α mRNA, direct binding of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) to major histocompatibility complex (MHC) class II molecules results in a fast (peak 1 h after stimulation), transient induction (sevenfold) of TNF-α mRNA. This induction correlates with a fourfold increase in transcription rates of the TNF-α gene, as detected by run-on assays, and does not require de novo protein synthesis. Mapping of DNase-I hypersensitive sites (DHS) discloses two constitutive DHS, one located far upstream (within the TNF-β promoter) and the other centered at −39 ± 40 bp relative to the major TNF-α transcription start site, suggesting that the TNF-α gene was transcriptionally competent even prior to MHC class II engagement. Furthermore, stimulation of human monocytes with either TSST-1 or lipopolysaccharide increases the translational efficiency of TNF-α mRNA, as shown by a shift in the distribution of this mRNA species in polysome gradients and the translation rates of TNF-α measured by immunoprecipitation from cells pulsed with [35S] methionine. The increase in translation efficiency of TNF-α mRNA is independent of the half-life of TNF-α transcripts, which under the conditions used is unchanged. Taken together, our data indicate that TNF-α expression is tightly regulated by MHC class II ligands, both at the transcriptional and translational levels.