The glucocorticoid receptor (GR) and c-jun promoters both contain activator protein-1 (AP-1) sites (GR AP-1 site and c-jun AP-1 site, respectively) that vary from the consensus AP-1 site. Electrophoretic mobility shift assays (EMSAs) were used to monitor GR AP-1 and c-jun AP-1 oligonucleotide binding by nuclear extracts from AtT-20 and L929 cells that were hormone- and vehicle-treated for 1, 6, or 24 h. Both AtT-20 and L929 cell nuclear extracts bound the c-jun AP-1 site somewhat better than the GRAP-1 site and, in the majority of cases, extracts from hormone-treated cells shifted both GRAP-1 and c-jun AP-1 oligonucleotides more than nontreated nuclear extracts. Supershift assays, using Jun and Fos family member-specific antibodies, showed that protein complexes formed by AtT-20 cell nuclear extracts bound to the c-jun AP-1 site were comprised of Jun family members, JunD, JunB, and cJun. No Fos family members were present. However, protein complexes from AtT-20 nuclear extracts that bound the GR AP-1 site were supershifted by JunD, JunB, cJun, and Fra-2 specific antibodies. In L929 cell nuclear extracts, the c-jun AP-1 site is bound by JunD and cJun. No clear association of Fos family members with the c-jun AP-1 site could be demonstrated. The GR AP-1 site bound protein complexes composed of JunD, JunB, Fra-2, and Fra-1 from L929 nuclear extracts. This demonstrates that the composition of the protein complexes that associate with the c-jun AP-1 site differs from those that bind the GR AP-1 site. These data also indicate that the protein complexes that bind the GR and c-jun AP-1 sites are cell-type-specific. Computer analysis also revealed five putative cyclic AMP response elements (CREs) in the GR promoter. Relative mobility shift and binding studies suggest that CRE binding protein (CREB), CREB modulator (CREM), or CREB/CREM may be associated with the c-jun AP-1 and/or GR AP-1 sites, but the association at these sites occurs at a lower binding affinity than for a consensus CRE. Nuclear extracts from AtT-20 and L929 cells were able to shift the CRE, and supershift analysis revealed that Jun family members are part of the protein complexes that bind the CRE. Pan Jun and pan Fos antibodies were able to supershift protein-CRE complexes formed using NIH 3T3 nuclear extracts. These data raise the possibility that the promiscuous binding of CREB and/or CREM to the AP-1 site, and AP-1 transcription factors to one or more CREs, in the GR promoter may contribute to the regulation of GR gene expression.