The activity of the AF-2 transcriptional activation function of nuclear receptors (NR) is mediated by the partially homologous transcriptional coactivators, glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1). GRIP1 and SRC-1 bound nine different NRs and exhibited similar, but not identical, NR binding preferences. The most striking difference was seen with the androgen receptor, which bound well to GRIP1 but poorly to SRC-1. GRIP1 and SRC-1 contain three copies of the NR binding motif LXXLL (called an NR Box) in their central regions. Mutation of both NR Box II and NR Box III in GRIP1 almost completely eliminated functional and binding interactions with NRs, indicating that these two sites are crucial for most of GRIP1's NR binding activity. Interactions of GRIP1 with the estrogen receptor were more strongly affected by mutations in NR Box II, whereas interactions with the androgen receptor and glucocorticoid receptor were more strongly affected by NR Box III mutations. One isoform of SRC-1 has an additional NR Box (NR Box IV) at its extreme C terminus with an NR-binding preference somewhat different from that of the central NR-binding domain of SRC-1. GRIP1 has no NR Box in its C-terminal region and therefore no C-terminal NR-binding function. In summary, GRIP1 and SRC-1 have overlapping NR-binding preferences, but specific NRs display both coactivator and NR Box preferences that may contribute to the specificity of hormonal responses.