We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf‐5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the β‐galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf‐5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf‐5/nlacZ transgenes. Thus, in a comparison using ES cells in chiameric embryos containing the targeted or randomly integrated myf‐5/nlacZ construct, we demonstrate that 5.5 kbp of myf‐5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf‐5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows β‐galactosidase activity, even after birth, indicating that myf‐5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf‐5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf‐5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice. © 1996 Wiley‐Liss, Inc.