Myf-5, a member of the family of four muscle-specific basic helix-loop-helix (bHLH) transcription factors is the first to be expressed in somites, branchial arches, and limb buds during prenatal mouse development. However, little is known about control mechanisms which actually regulate Myf-5 gene activity within these various muscle-forming domains. To identify control regions that contribute to the correct spatiotemporal activity pattern of the Myf-5 gene during mouse embryogenesis, here we report the characterization of yeast artificial chromosomes (YACs) which faithfully direct muscle-specific expression of the gene in chimeric mouse embryos. Forty-five kilobases of sequence 5' to the Myf-5 gene together with 500 kb of 3' flanking DNA drives the correct Myf-5 expression in the mesenchyme of the visceral arches and in somites but not in the hypaxial muscles of limb buds. An additional 50 kb of DNA at the 5' end is required to activate Myf-5 gene expression in developing limbs. These results demonstrate for the first time that unexpectedly distant regions of the Myf-5 gene are necessary to recapitulate its precise developmental expression pattern. We also show that Myf-5 expression in hypaxial muscles and in somites and visceral arches is regulated by separate and distinct far upstream regions. The identification of these remote regulatory elements on YACs carrying the mouse Myf-5 gene constitutes the first important step toward further dissection of the complex mechanisms by which cell-autonomous and external cues control Myf-5 expression during skeletal muscle formation in the mouse embryo.