The term AP-1 describes homodimeric and heterodimeric transcription factors composed of members of the Jun, Fos and CREB/ATF families of proteins. Distinct AP-1 dimers, like for instance the prototypical c-Jun:c-Fos and c-Jun:ATF2 dimers, are differentially regulated by signaling pathways, and bind related yet distinct response elements in the regulatory regions of AP-1 target genes. Little is known about the dimer specific regulation of AP-1 activity at the promoter of its target genes. We have previously shown that nTrip6, the nuclear isoform of the LIM domain protein Trip6, acts as an AP-1 co-activator. Moreover, nTrip6 is an essential component of glucocorticoid receptor (GR)-mediated trans-repression of AP-1, in that it mediates the tethering of GR to the promoter-bound AP-1. We now discovered a striking specificity of nTrip6 actions determined by the binding preference of its LIM domains. We show that nTrip6 interacts only with Fos family members. Consequently, nTrip6 is a selective co-activator for AP-1 dimers containing Fos. nTrip6 also assembles activated GR to c-Jun:c-Fos-driven promoters. Neither nTrip6 nor GR are recruited to a promoter occupied by c-Jun:ATF2. Thus, only Fos containing dimers are trans-repressed by GR. Thus, the dimer composition of AP-1 determines the mechanism of both the positive and negative regulation of AP-1 transcriptional activity. Interestingly, on a second level of action, GR represses the increase in transcriptional activity of c-Jun:ATF2 induced by JNK-dependent phosphorylation. This repression depends on GR-mediated induction of MAP kinase phosphatase 1 (MKP-1) expression, which results in JNK inactivation.