This study is the first to report the comprehensive analysis of Ank3 expression and alternative splicing in the heart. We demonstrate that the heart expresses multiple ankyrin-G isoforms and that ankyrin-G isoforms are detected at the intercalated discs and T-tubules of individually isolated cardiomyocytes. Using a PCR-based screen of cardiac mRNA, we identify two new exons in the Ank3 gene and 28 novel splicing events in Ank3 transcripts. We measure the relative ventricular expression of each HCV-796 splice junction by quantitative OTSSP167 real-time PCR with transcript-specific primers. We demonstrate that expression of exon 1d, one of the five first Ank3 exons, is restricted to heart and skeletal muscle. We evaluate some of the alternative splice isoforms for altered function and find that two rare isoforms of the ankyrin-G spectrin binding domain lack spectrin binding. In summary, this study demonstrates that the Ank3 gene is subject to complex splicing regulation resulting in numerous ankyrin-G isoforms in heart. We anticipate that these different isoforms underlie the diversity of ankyrin-G functions and subcellular distribution within cardiomyocytes. This study is the first to describe a comprehensive analysis of Ank3 expression in heart. We identified two new exons and 28novel alternative splicing events in the Ank3 gene. An update to the Ank3 exon organization and nomenclature is provided in Table2. Using quantitative real-time PCR with exon-exon spanning primers, we demonstrate that alternative Ank3 splice variants are expressed at similar levels in three separate hearts. Moreover, we find that expression of Ank3 transcripts initiated with exon disrestricted to the heart and skeletal muscle as these transcripts are undetectable in brain, kidney, cerebellum, and lung. The majority of alternative splicing is situated within the coding region of the spectrin binding domain, specifically within the exons immediately 5�� of the minimal spectrin-binding domain ZU5A, which is encoded by exons 31 and 32.The newly identified exons 27 and 30are also alternatively spliced, but neither exon alters the open-reading frame of ankyrin-G and exon 30 is rarely expressed in cardiac transcripts. Interestingly, exon 30 is included in three expressed sequence tags that were isolated from neural tissue.