We report glucocorticoid-dependent induction of transcription from the herpes simplex virus thymidine kinase gene promoter proximal regulatory region in the absence of glucocorticoid response elements and independent of the ability of glucocorticoid receptor (GR) to bind DNA. Examination of the thymidine kinase promoter localized glucocorticoid responsiveness to a binding site for CCAAT enhancer-binding proteins (C/EBPs). Further analysis indicated that GR specifically potentiated the induction of transcription by C/EBPβ, but not C/EBPα or δ, and that full induction could be obtained by the ligand-binding domain (LBD) of GR alone. C/EBPβ, but not C/EBPα or δ, reciprocally potentiated transcriptional activation by DNA-bound GR LBD. However, C/EBPβ was unable to increase activation by a GR LBD with a short C-terminal truncation, indicating that the functional interaction between the two factors was dependent upon the GR AF-2. Surprisingly, despite the specificity in functional effects, all three C/EBPs bound indistinguishably to GR in GST pull-down and immunoprecipitation assays. Indeed, several nuclear receptors, including the estrogen (ERα), progesterone, retinoic acid (RAR), and androgen receptors, displayed a similar potential to bind C/EBPs. Previous reports have demonstrated that ERα and RARs repress transcriptional activation by C/EBPβ in ways that were dependent on their related AF-2 functions. Therefore, the GR AF-2 may encode functional features that distinguish the transcriptional regulatory potential of GR from that of ER and RAR. Finally, C/EBP binding mapped to the GR DNA-binding domain, which was not required for functional interaction with C/EBPβ. Thus, the potentiation of C/EBPβ-mediated transcription by GR would appear to require the presence of an intermediary factor.