While the biochemical mechanisms mediating repression of NF-kappaB activity by glucocorticoids (GCs) are relatively well studied, the role of promoter architecture for the effects of GCs on NF-kappaB remains poorly characterized. Therefore we constructed a set of synthetic promoter reporter constructs containing various numbers of GC-responsive elements (GREs) in distinct distances to NF-kappaB binding sites. TNFalpha-induced activity of a synthetic promoter controlled by three NF-kappaB binding sites was repressed by dexamethasone. The presence of only one GRE in the vicinity of the KB sites abolished this repression and allowed synergistic transcriptional activation by NF-kappaB and the glucocorticoid receptor (GR). The synergism identified here was not affected by the number of GREs, but strictly depends on the spacing between GREs and KB sites. These experiments reveal that the functional interplay between NF-kappaB and the GR also involves--dependent on the promoter context--synergistic stimulation of transcription.