The complete exon-intron organization of the murine gene encoding M-protein, a structural protein of sarcomeric myofibrils, was determined. The gene is composed of 37 exons and 36 introns, spanning approximately 75 kb of DNA. Intron positions are related to the modular structure of M-protein, which is composed essentially of immunoglobulin and fibronectin type III domains. Almost all repeats follow a two exon-one domain structure. The beginning and end of each domain are defined by introns in phase I; internal introns are more divergent in position and very rarely use phase I. A single transcriptional start point was detected in both skeletal and cardiac muscle. Analysis of the prospective promoter region revealed several potential regulatory elements. CAT expression assays using promoter deletion constructs identified three regions that seem to be most important for the muscle-specific transcription activation of the M-protein gene. These results provide the first complete characterization of a gene for a member of the intracellular branch of the immunoglobulin superfamily.