PEA-15 is a small anti-apoptotic protein that is enriched in astrocytes, but expressed in a broad range of tissues. It sequesters the protein kinases ERK1 and 2 in the cytoplasm, thereby limiting their proximity to nuclear substrates. Using a fluorescence anisotropy approach, PEA-15 is shown to be a high-affinity ligand for both ERK1 and 2, exhibiting a dissociation constant in the range of Kd = 0.2-0.4 microM, regardless of their activation states. Neither the phosphorylation of PEA-15 (phospho Ser-104 and/or phospho Ser-116) nor the phosphorylation of ERK1/2 (by MKK1) significantly affects the stability of the ERK/PEA-15 interaction, and therefore it does not directly regulate the release of ERK2 to the nucleus. The extreme C-terminus of PEA-15 was previously shown by mutagenesis to be important for ERK2 binding; however, the site of binding was not established. Here it is demonstrated that the D-recruitment site (DRS) of ERK2 binds PEA-15, probably at the C-terminus, and renders PEA-15 an inhibitor of ERK2 docking interactions. Using fluorescence anisotropy competition assays it is shown that PEA-15 competes for binding to ERK1/2 with a peptide derived from the D-site of Elk-1, which binds the DRS of ERK1/2. Using modified ERK2 proteins containing single cysteine residues, PEA-15 was shown to protect single cysteines situated within the DRS from alkylation. The pattern and magnitude of protection were very similar to those induced by the binding of the peptide derived from the D-site of Elk-1. These and published data support the notion that PEA-15 binds two sites on ERK1/2 in a bidentate manner: the DRS and a site that includes the MAP kinase insert. Previous reports have suggested that PEA-15 is not an inhibitor of ERK2; however, it is shown here to potently inhibit the ability of ERK2 to phosphorylate two transcription factors, Elk-1 and Ets-1, which contain docking sites for the DRS of ERK2. Therefore, in addition to sequestering ERK1/2 in the cytoplasm, PEA-15 has the potential to modulate the activity of ERK2 in cells by competing directly with proteins that contain D-sites.