The transcription factors Serum Response Factor (SRF) and Ternary Complex Factor (TCF) form a ternary complex at the c‐fos Serum Response Element (SRE). We show that in NIH3T3 cells TCF binding is required for regulated transcription in response to stimulation by phorbol myristate acetate (PMA), but not by whole serum. We constructed a novel transcriptionally inactive SRE variant whose serum‐regulated activity can be partially restored by overexpression of SRF in the absence of bound TCF. Activation by SRF does not require the SRF N‐terminal phosphorylation sites, but is potentiated 2‐ to 3‐fold by the SRF C‐terminal activation domain. Mutations in the SRF DNA binding domain, which do not affect the ability of SRF to bind DNA, abolish its ability to mediate TCF‐independent serum‐regulated activation and reduce activation by the SRF/TCF(Elk‐1) ternary complex. Efficient activation requires that SRF be targeted to DNA via its own DNA binding domain.