In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O-MACS, a member of the medium-chain acyl-CoA synthetase family. The o-macs gene encodes a protein of 580 amino acids, sharing 56-63% identity with other MACS family proteins. RT-PCR analysis demonstrated that the o-macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o-macs transcripts are present in the neuronal cell layer of olfactory sensory neurons (OSNs) as well as in the supporting and basal cell layers in the most dorso-medial area (zone 1) of the OE. Developmental analysis revealed that the o-macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O-MACS protein tagged with c-Myc and His6 demonstrated an acyl-CoA synthetase activity for fatty acid activation, and protein localization to mitochondria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development.