Follistatin is a secreted protein, which functions as an antagonist of different members of the TGF-beta superfamily, including activin and bone morphogenetic proteins. Expression of follistatin is tightly regulated during mouse development both spatially and temporally. In order to study the regulation of follistatin expression in the mouse embryo we have cloned and analyzed part of the 5' flanking region of the murine follistatin gene. Primer extension and RNase protection assays demonstrate that the murine follistatin promoter region has at least three distinct transcription initiation sites, which are each preceded by a TATA box. All of the transcription initiation sites are located within the first 500 bp upstream of the translational start site. Sequence analysis of this 500 bp region revealed several consensus binding sites for transcription factors including AP-1, Brachyury-T, CREB, Sp1, AP-2 and Tcf. To test whether the 5' region displays promoter activity, we transfected various 5' flanking region deletion constructs into F9 embryonal carcinoma (EC) cells and into P19 EC cells. In these two cell lines a region of only 262 bp upstream of the translation start site could drivereporter expression in a manner that reflects endogenous mRNA expression.