FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development.