The Elk-l and SRF transcription factors form a ternary complex at the c-fos serum response element (SRE). Growth factor stimulation rapidly induces a reversible change in the electrophoretic mobility of the ternary complex, accompanied by increased phosphorylation of the Elk-i C-terminal region and by the activation of a 42 kd cellular Elk-l kinase. Phosphorylation of Elk-l in vitro by partially purified p42/p44 MAP kinase induces a similar reduction in ternary complex mobility but has little effect on the eff iciency of its formation. In vitro, MAP kinase phosphorylates the Elk-l C-terminal region at multiple sites, which are also phosphorylated following growth factor stimulation in vivo. The Elk-l C-terminal region functions as a regulated transcriptional activation domain whose activity in vivo is dependent on the integrity of the MAP kinase sites. These findings directly link transcriptional activation by the SRE to the growth factor-regulated phosphotylation of an SRE-binding protein.