Serum response factor (SRF) regulates transcription of many serum-inducible and muscle-specific genes. Using a functional screen, we identified LIM kinase-1 as a potent activator of SRF. We show that SRF activation by LIM kinase-1 is dependent on its ability to regulate actin treadmilling. LIM kinase activity is not essential for SRF activation by serum, but signals depend on alterations in actin dynamics. Studies with actin-binding drugs, the actin-specific C2 toxin, and actin overexpression demonstrate that G-actin level controls SRF. Regulation of actin dynamics is necessary for serum induction of a subset of SRF target genes, including vinculin, cytoskeletal actin, and srf itself, and also suffices for their activation. Actin treadmilling provides a convergence point for both serum- and LIM kinase-1-induced signaling to SRF.