Glucocorticoid (GC) effects are mediated via the GC-receptor (GR), which either stimulates or represses gene expression. Repression of target genes often involves negative cross-talk between the GR and other transcription factors e.g. NF-kappaB, important for gene activation. Using HEK293 cells we here describe that repression of NF-kappaB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-kappaB. While a GR mutant was able to repress NF-kappaB activity following activation by TNFalpha, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFalpha, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-kappaB activity was restored in conjunction with ERK1/2 inhibition. Previous reports have shown GC-mediated inhibition of ERK1/2 phosphorylation to involve GC induction of MAPK phosphatase-1 (MKP-1). Here, we demonstrated that the GRR488Q mutant was incapable of inducing gene expression of endogenous MKP-1 following dexamethasone treatment, in contrast to the GRwt. However, TPA treatment alone resulted in much stronger MKP-1 expression in both GRwt and GRR488Q containing cells than that of dexamethasone suggesting that the inability of GRR488Q to inhibit TPA-induced NF-kappaB activity did not involve a lack of MKP-1 expression. In line with this, RNAi targeted towards MKP-1 did not abolish or inhibit the ability of the GRwt to repress NF-kappaB activity. Importantly, we observed no difference in activated ERK1/2 (phospho-ERK1/2) expression over time between GRwt and GRR488Q containing cells following co-treatment with TPA and dexamethasone. Based on these results we suggest that GRwt does not directly regulate ERK1/2 but rather alters ERK1/2-mediated effects allowing it to repress NF-kappaB activity, a capacity lacked by the GRR488Q mutant.