Recruitment of multipotent mesodermal cells to the myogenic lineage is mediated by the transcription factor Myf5, the first of the myogenic regulatory factors to be expressed in most sites of myogenesis in the mouse embryo. Among numerous elements controlling the spatiotemporal pattern of Myf5 expression, the -58/-56 kb distal Myf5 enhancer directs expression in myogenic progenitor cells in limbs and in somites. Here, we show by site-directed mutagenesis within this enhancer that a predicted homeobox adjacent to a putative paired domain-binding site is required for the activity in muscle precursor cells in limbs and strongly contributes to expression in somites. By contrast, predicted binding sites for Tcf/Lef, Mef3 and Smad transcription factors play no apparent role for the expression in limbs but might participate in the control in somites. A 30mer oligonucleotide sequence containing and surrounding the homeo and paired domain-binding motifs directs faithful expression in myogenic cells in limbs and also enhances myotomal expression in somites. Pax3 and Meox2 transcription factors can bind to these consensus sites in vitro and therefore constitute potential regulators. However, genetic evidence in the Meox2-deficient mouse mutant argues against a role for Meox2 in the regulation of Myf5 expression. The data presented here demonstrate that a composite homeo and paired domain-binding motif within the -58/-56 enhancer is required and sufficient for activation of the Myf5 gene in muscle progenitor cells in the limb. Although Pax3 constitutes a potential cognate transcription factor for the enhancer, it fails to transactivate the site in transfection experiments.