Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRF⌬5 to SRF. Increased levels of SRF⌬5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRF⌬5 variant relative to wild-type SRF. SRF⌬5 forms DNA binding-competent homodimers and heterodimers. SRF⌬5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal ␣-actin, cardiac ␣-actin, smooth ␣-actin, SM22␣, and SRF promoter-luciferase reporter activities. Expression of SRF⌬5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal ␣-actin and myogenin mRNAs. SRF⌬5 repressed the serum-induced activity of the c-fos serum response element. SRF⌬5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRF⌬5, may play an important regulatory role in modulating SRF-dependent gene expression.Alternative splicing is a commonly used molecular strategy for creating diverse gene products from a single genetic locus in most eucaryotic cells. The modular organization of transcription factor genes, through exon-encoded structural domains, may be conducive for forming a variety of alternatively spliced isoforms that affect DNA binding avidity and specificity, transactivation, subcellular localization, responsiveness to signaling pathways, and developmental regulation (reviewed in reference 31). For example, alternative splicing within the DNA binding domain of Pax-6 (9) and Wilm's tumor-associated protein 1 (5, 21) alters their DNA binding specificities. Alternative splicing of exons encoding the transactivating domains in the paired family proteins Pax-3 (59), , and Pax-9 (39), the POU homeodomain family proteins Pit-1 (37), Oct-1 (8), Oct-2 (1), and Brn-3a (38), and the zinc finger transcription factor GATA-5 (32) results in isoforms possessing activation domains with different potencies. Splicing out of select exons from the activation domain as in AML1a (56), Oct-2 (29), and CREB family proteins CREB (reviewed in reference 11), Drosophila CREB/CREM (61), and CREM (12) produces isoforms with dominant negative activity. Similarly, several splice variants containing only the DNA binding domain act as dominant negative isoforms presumably by heterodimerizing w...