After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.