Extraocular muscle (EOM) exhibits high-velocity, low-tension contractions compared with other vertebrate striated muscles. These distinctive properties have been associated with a novel myosin heavy chain (MyHC) isoform, MyHC-EO. An atypical MyHC, MyHC IIL, has also been identified in laryngeal muscles that have similarly fast contractile properties. It co-migrates with MyHC-EO on high-resolution SDS gels, but appeared to be encoded by a different mRNA. We combined CNBr peptide maps and full-length cDNA sequences to show that rabbit muscle EO and IIL MyHCs are identical. Analysis of the 5; untranslated region (5;UTR) of the mRNAs identified three variants that result from a combination of alternative splicing and multiple transcription initiation sites. This complex pattern of 5;UTRs has not been reported previously for MyHC genes. We identified the human homologue of the MyHC-EO gene in GenBank, and analyzed the 5; upstream region, which revealed a paucity of muscle-specific transcription factor binding sites compared with the other MyHC genes. These features are likely to be critical to the unique regulation and tissue-specific expression of the MyHC-EO/IIL gene. Phylogenetic analysis indicates that MyHC-EO/IIL diverged from an ancestral MyHC gene to generate the first specialized fast myosin. The catalytic S1 head domain is more closely related to the fast MyHCs, while the rod is more closely related to the slow/cardiac MyHCs. The exon boundaries of the MyHC-EO are identical to those of the embryonic MyHC gene and virtually identical to those of the &agr; and (&bgr;) cardiac genes. This implies that most of the current exon boundaries were present in the ancestral gene, predating the duplications that generated the family of skeletal and cardiac myosin genes.