The serum response factor (SRF) is essential for embryonic development and maintenance of muscle cells and neurons. The mechanism by which this factor controls these divergent pathways is unclear. Here we present a genome-wide view of occupancy of SRF at its binding sites with a focus on those that vary with cell type. We used chromatin immunoprecipitation (ChIP) in combination with human promoter microarrays to identify 216 putative SRF binding sites in the human genome. We performed independent quantitative PCR validation at over half of these sites that resulted in 146 sites we assert to be true binding sites at over 90% confidence. Nearly half of the sites are bound by SRF in only one of the three cell types we tested, providing strong evidence for the diverse roles for SRF in different cell types. We also explore possible mechanisms controlling differential binding of SRF in these cell types by assaying cofactor binding, DNA methylation, histone methylation, and histone acetylation at a subset of sites bound preferentially in smooth muscle cells. Although we did not see a strong correlation between SRF binding and epigenetics modifications, at these sites, we propose that SRF cofactors may play an important role in determining cell-dependent SRF binding sites. ELK4 (previously known as SAP-1 [SRF-associated protein-1]) is ubiquitously expressed. Therefore, we expected it to occupy sites where SRF binding is common in all cell types. Indeed, 90% of SRF sites also bound by ELK4 were common to all three cell types. Together, our data provide a more complete understanding of the regulatory network controlled by SRF. ©2007 by Cold Spring Harbor Laboratory Press.