Myogenesis of skeletal muscles in vertebrates is controlled by extracellular signalling molecules together with intracellular transcription factors. Among the transcriptional factors, the members of the myogenic regulatory family, including MyoD, Myf5, Myogenin and MRF4, play important roles regulating skeletal muscle development and growth. To characterize the gene structure and expression of fish myogenin, we have isolated the myogenin genomic gene and cDNA from gilthead seabream (Sparus aurata) and analyzed the genomic structure, pattern of expression and the regulation of muscle-specific expression. Sequence analysis revealed that the seabream myogenin shares a similar gene structure with other fish myogenins, with three exons, two introns and the highly conserved bHLH domain. Expression studies demonstrated that myogenin is expressed in both slow and fast muscles as well as in muscle cells in primary culture. In situ hybridization showed that myogenin was specifically expressed in developing somites of seabream embryos. Promoter activity analysis demonstrated that the myogenin promoter could drive green fluorescence protein expression in muscle cells of zebrafish embryos, as well as in myofibers of adult zebrafish and juvenile seabream. Deletion analysis demonstrated that this muscle-specific activity depends on the presence of a MEF2 and a MEF3 binding site within the 550 bp myogenin promoter sequence.