Serum response factor (SRF) has been shown to play a key role in cardiac cell growth and muscle gene regulation. To understand the role of SRF in heart failure, we compared its expression pattern between control and failing human heart samples. Western blot analysis of control samples showed expression of four different isoforms of SRF, with ∼67-kDa full-length SRF being the predominant isoform. Interestingly, in failing hearts we found robust expression of a low-molecular-mass (∼52 kDa) SRF isoform, accompanied by decreased expression of full-length SRF. By RT-PCR and Southern blot analyses, we characterized this ∼52-kDa SRF isoform as being encoded by an alternatively spliced form of SRF lacking exons 4 and 5 of the SRF primary RNA transcript (SRF-Δ4,5 isoform). We cloned SRF-Δ4,5 cDNA and showed that overexpression of this isoform into cells inhibits SRF-dependent activation of cardiac muscle genes. These results suggest that expression of SRF-Δ4,5 in failing hearts may in part contribute to impaired cardiac gene expression and consequently to the pathogenesis of heart failure.