Serum response factor (SRF) is a transcription factor that binds the sequence CC(A/T)6GG found in a number of growth factor-inducible and muscle-specific promoters. We describe the isolation and characterization of cDNA clones encoding a family of three human SRF-related DNA-binding proteins. Each of these RSRF (related to SRF) proteins contains an 86-amino-acid amino-terminal region related to the SRF DNA-binding domain: In RSRFC4 and RSRFC9, this region is identical, whereas that present in RSRFR2 differs by seven conservative amino acid substitutions. The DNA-binding specificity of the RSRF proteins, which recognize the consensus sequence CTA(A/T)4TAG, is distinct from that of SRF. The entire RSRF common region is required for DNA binding, and the differential sequence specificity of the RSRFs and SRF is the result of differences in the basic amino-terminal part of this domain. The RSRF proteins bind DNA as dimers and can dimerize with one another but not with SRF. Although the RSRF mRNAs are expressed in many cell types, RSRFR2 mRNA is expressed at elevated levels in several B-cell lines. Consistent with this, extracts from many cell types form CTA(A/T)4TAG-binding complexes that contain RSRF proteins, and oligonucleotides containing RSRF-binding sites function as promoter elements in transfection assays. Like SRF-binding sites, RSRF-binding sites are found in the regulatory sequences of a number of growth factor-inducible and muscle-specific genes, and we show that RSRF polypeptides are components of previously characterized binding activities that interact with these elements. We discuss the potential role of RSRF proteins in the regulation of these genes.