Immunocytochemical analysis has demonstrated that expression of the olfactory marker protein (OMP) is highly restricted to mature olfactory receptor neurons in virtually all vertebrate species from fish to man. We have now cloned the OMP gene from human and mouse and demonstrated conservation of gene structure, protein sequence, and Olf-1 and upstream binding region (UBE) regulatory domains. The OMP gene in all species studied lacks canonical TATA and CAAT motifs and introns. The deduced protein sequence is 88.4% identical between mouse and human, and most of the differences observed are conservative changes. The proximal Olf-1 binding sites differ by two purine-purine replacements and effectively cross-compete in mobility shift assays. The distal Olf-1 binding site is also highly conserved in terms of both sequence and binding activity. The availability of sequence from multiple species has permitted us to determine that the UBE site has close similarity to motifs that bind members of the NF-1 family of transcription factors. Gel mobility shift assays confirm this prediction, providing additional insight into mechanisms that may participate in the stringent regulation of the expression of this neuronal-specific protein. Furthermore, we demonstrate the in situ localization of OMP mRNA in human olfactory neuro-epithelium and its colocalization to immunocytochemically identified human olfactory receptor neurons.