Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (ME) with an accessory factor, Elk-l. We constructed altered-binding specificity derivatives of SRF and Elk-l that form a ternary complex at a mutated, inactive SRE; like Elk-l, the Elk-l variant only binds its target as part of a ternary complex with SRF. Simultaneous expression of these SRF and Elk-l derivatives restores serumregulated activity to the mutated SRE in transfected cells. Efficient transcriptional activation is dependent on the regulated phosphorylation of Elk-l C-terminal MAP kinase sites and requires the C-terminal sequences of SRF as well as SRF sequences that mediate ternary complex formation. These experiments provide direct evidence that SRF and Elk-l functionally cooperate in the ternary complex at the SRE to regulate transcription.