Glucocorticoid administration and/or excess secretion have been associated with increased Human Immunodeficiency Virus Type-1 (HIV-1) replication and AIDS progression. The HIV-1 long terminal repeat (LTR) promoter contains glucocorticoid-responsive element (GRE)-like sequences that could mediate a positive effect of glucocorticoids on HIV-1. In addition, we recently demonstrated that the HIV-1 accessory protein Vpr is a potent coactivator of the glucocorticoid receptor, which, like the host coactivator p300, potentiates the effect of glucocorticoids on GRE-containing, glucocorticoid-responsive genes. Such an effect may increase the sensitivity of several host target tissues to glucocorticoids by several fold, and may, thus, contribute to a positive effect of glucocorticoids on the HIV-1-LTR in infected host cells. In this study, we determined the direct effect of glucocorticoids on HIV-1-LTR by examining the ability of dexamethasone to modulate the activity of this promoter coupled to the luciferase reporter gene in human cell lines. Dexamethasone markedly inhibited Tat-stimulated, p300- or Vpr-enhanced luciferase activities in a cell-type specific, dose-dependent, and glucocorticoid receptor-mediated fashion. This effect of dexamethasone was not potentiated by Vpr, was antagonized by the glucocorticoid receptor antagonist RU 486 and required the DNA-binding domain of the receptor. These data suggest that the inhibitory effect of glucocorticoids on the HIV-1-LTR may be exerted via non-GRE-dependent inhibition of the strongly positive host transcription factor NF-kappaB, which interacts with the DNA- and ligand-binding domains of the receptor. Alternatively, it is also possible that dexamethasone-activated glucocorticoid receptor competes with other transcription factors for their binding sites on the promoter region or squelches transcription factors shared by HIV-1-LTR and glucocorticoid-responsive promoters. We conclude that glucocorticoids suppress, rather than stimulate, the HIV-1 promoter, thus acting, protectively for the host. Their apparent negative clinical association with AIDS is most likely due to immunosuppression of the host.