Coactivators of nuclear receptors are integral components of the signal transduction pathways of steroid hormones. Here, we show that one of the major coactivators of the glucocorticoid receptor (GR), CREB-binding protein (CBP), can also function conditionally as a negative regulator of its activities. Indeed, CBP suppressed the responsiveness of the mouse mammary tumor virus (MMTV) promoter to dexamethasone in a dose-dependent fashion in HeLa and A204 cells. Similarly, this protein suppressed the responsiveness of several glucocorticoid-responsive element (GRE)-containing synthetic promoters. Using deletion mutants of CBP, we localized the repressor effect of this protein to its N-terminal domain and showed that it was independent of the histone acetyltransferase and coactivator-binding domains but dependent upon its GR-binding domain. We also demonstrated functional differentiation between CBP and other coactivators, including SRC-1 and the CBP-related protein p300, both of which influenced GR signaling in a positive fashion. In fact, p300 completely antagonized the suppressive effects of CBP in a dose-dependent fashion, probably by competing with this protein at the level of the transcription complex. These findings suggest that CBP and p300 may function additively or antagonistically to each other depending on their relative concentrations and type of target tissue, to influence the sensitivity of tissues to glucocorticoids.