Ternary complex factors (TCFs), one of which is Elk-1, have been implicated in mediation of c-fos induction. They have been shown to be phosphorylated by mitogen-activated protein kinases (MAPKs) in vitro. We demonstrate that recombinant Elk-1 is hyperphosphorylated in vivo upon joint overexpression of MAPKs and constitutively activated Raf-1 kinase, the latter serving as an indirect in vivo activator of MAPKs. This phosphorylation is accompanied by a conformational change and results in an elevated transactivation potential of Elk-1. Mutation of mapped in vivo phosphorylation sites, which are potential targets for MAPKs, reduced Elk-1-mediated transcription. Thus, MAPKs are very probably controlling Elk-1 activity by direct phosphorylation in vivo. Furthermore, Elk-1 was shown to stimulate transcription from both the c-fos serum response element and also from an Ets binding site. While binding of TCFs to the c-fos promoter is dependent on the serum response factor, TCFs can autonomously interact with Ets binding sites. This indicates that TCFs may participate in the transcriptional regulation of two different sets of genes.