Among the transducers of the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) receptor signaling proteins, Smad4 and Smad1 act together in BMP 2/4 signaling pathways, and Smad4 and Smad2 act in TGF-beta/activin signaling. To investigate how the Smad4 gene is regulated at the transcriptional level, we cloned and characterized its 5'-flanking region. The major transcription start site mapped by primer extension analysis was 132 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes; it did, however, contain a TATA-like structure (TAAAAT) 32 bp upstream of the transcription start site. A consensus sequence for homeoprotein HoxA-5 (TTTAAAAATTA) was identified at 171 bp upstream of the transcription start site. Within 600 bp upstream of the transcription start site, two Pit-1 and four F2F binding sites were found. One putative AP-1 site was located at -1129. These findings suggest that these homeoproteins could conduct their signals specifically by controlling both inter- and intracellular signal transduction pathways.