Mix/Bix proteins represent a vertebrate subgroup of paired-like homeodomain proteins which are known to function around the time of gastrulation. Here we report the structures of the genomic and upstream promoter regions of a mouse and human Mix-like gene. Both genes map to syntenic regions of chromosome 1 and contain two coding exons, with the paired-type homeodomain split between the exons within helix 3. Differentiating mouse embryonic stem cells transcribe a messenger RNA of approximately 2.6 kb. The first exon encodes the translation initiation codon and a 5' untranslated region of approximately 90 bp. Sequence analysis of the 960 bp upstream of the transcription start site of the mouse Mix gene revealed the presence of a putative initiator region and TATA box as well as potential Smad, FoxH1/FAST, T-box, COUP-TF, C/EBP, GATA, HNF3 binding sites and retinoic acid response elements. A number of these sites are conserved in the human Mix promoter. We find that most paired-related homeodomain proteins, including mouse and human Mix, contain a proline-rich region within their amino termini which may interact with other proteins. Mouse and human Mix proteins contain highly conserved carboxy-terminal polar/acidic regions with the potential to form an amphipathic helix and the ability to activate transcription in yeast. Mouse Mix expressed in COS cells or in vitro binds a DNA consensus sequence identified previously for paired class homeodomain proteins. These studies suggest that a number of features of paired-like protein structure and function are conserved across diverse species and provide a useful framework for studying the function and regulation of the mouse Mix gene.