We have analyzed nearly 2,000 myosin heavy chain gene (Myh) clones representing over 30 different transcripts from seven of eight striated muscle Myh genes expressed in mouse. We also report the transcriptional start sites (TSS) for the mouse developmental Myh genes. The data reveal a previously unknown diversity of TSSs and 5'-end alternative splicing in these transcripts. The cardiac Myh6 gene had two major TSSs. Use of the major downstream site led to an alternatively spliced second exon. Each of the other Myh genes had one major TATA-directed TSS and one or more minor alternative TSSs, some associated with alternative splicing. The minor transcripts were associated with polysomes and their spatial-temporal expression largely mirrored that of the major transcripts in wild-type, Myh1 null, Myh4 null, injured, and uninjured muscle, except that one form of Myh7, detected in heart, was not detected in diaphragm, and the ratio of the two major Myh6 transcripts varied in some circumstances. These findings indicate that alternative TSS usage and alternative splicing in the 5'-UTR are a general feature of murine Myh gene expression and that Myh gene regulation is more complex than previously appreciated.