Tumor necrosis factor (TNF) promotes certain immune and inflammatory responses, whereas glucocorticoids exert immunosuppressive and anti-inflammatory actions. We show that TNF treatment produced a modest inhibition of glucocorticoid receptor (GR)-mediated transcriptional activation of a mouse mammary tumor virus (MMTV) promoter-driven luciferase construct in HeLa cells. The mitogen-activated protein (MAP) kinases, p38 and c-Jun N-terminal kinase (JNK), are important mediators of target gene activation by TNF, and JNK activation was earlier shown to inhibit GR-mediated transcriptional activation by direct phosphorylation of GR at Ser-246. Transfection of HeLa cells with MKK6b(E), a constitutively active specific upstream activator of p38, led to a potent inhibition of GR activation of the MMTV promoter-driven luciferase construct. A similar inhibition of activation of the MMTV promoter-driven luciferase construct was seen in HeLa cells transfected with MKK7(D), a constitutively functional activator of JNK. Data from "domain swap" experiments using GR chimeras indicated that the main target of the p38-mediated (but not JNK-mediated) inhibition is the ligand-binding domain of GR (spanning amino acids 525-795), whereas the constitutively active N-terminal AF-1 region (spanning amino acids 106-237) is dispensable for the inhibitory effect of p38. We also demonstrate that activated p38 targets the GR ligand-binding domain indirectly. Suppression of GR function by activated p38 and JNK MAP kinases may be physiologically important as a mechanism of resistance to glucocorticoids seen in many patients with chronic inflammatory conditions.