The ETS transcription factor ER81 is activated in response to many signals via mitogen-activated protein kinases (MAPKs). However, ER81 is not only phosphorylated on MAPK sites but also at other sites that impact on its transactivation potential. Here we describe that the 90-kDa ribosomal S6 kinase 1 (RSK1), a protein kinase downstream of the extracellular signal-regulated kinase (ERK) subclass of MAPKs, binds to ER81, phosphorylates it, and enhances ER81-dependent transcription. Two in vivo RSK1 phosphorylation sites within ER81, Ser(191) and Ser(216), were identified, whose mutation to alanine reduces ER81 activity upon ERK-MAPK stimulation. Furthermore, RSK1 activates the ER81 cofactor CREB-binding protein and may thereby augment ER81-dependent transcription. Similar to RSK1, the cAMP-dependent protein kinase A (PKA) phosphorylates ER81 on Ser(191)/Ser(216). Additionally, PKA targets ER81 on Ser(334) in vivo. Surprisingly, phosphorylation of Ser(334) severely reduces the DNA-binding ability of ER81 but also enhances the transactivation potential of ER81. These counteractive effects of PKA phosphorylation on ER81-dependent transcription may cause the selective up-regulation of promoters with high but not low affinity for ER81. Collectively, we have identified mechanisms for how two distinct signaling pathways with different effector protein kinases, RSK1 and PKA, converge on ER81, which may regulate ER81 function during development and tumorigenesis.