Olfactory marker protein (OMP), previously thought to be expressed only by olfactory receptor neurons and their processes, was localized anatomically with immunocytochemical techniques to a number of brain regions in three rodent species, the mouse, rat, and hamster. In addition, the amount of antigen was quantified by radioimmunoassay (RIA) and characterized by an immunoblot procedure. In all three species the antigen could be detected immunocytochemically in the preoptic region and hypothalamus. The rat did not exhibit immunostaining in any other brain region. However, in the mouse neuronal labelling was observed throughout the neural axis, including cellular labelling in the bed nucleus of the anterior commissure, the median preoptic nucleus, the bed nucleus of the stria terminalis, the periventricular region, the anterior parvicellular subnucleus of the paraventricular nucleus, around the dorsomedial hypothalamic nucleus (pars compacta), the subincertal region, the arcuate nucleus, the anterior cortical nucleus of the amygdala, the suprageniculate nucleus, the lateral lemniscal nuclei, the lateraldorsal and lateralventral central gray, the posterior aspects of the commissural and marginal nuclei of the inferior colliculus, the paragenule nucleus, the A-5 region, the area postrema, the ventromedial nucleus of the solitary tract, area X, the spinal trigeminal nucleus (pars zonale), and superficial laminae of the spinal cord. The hamster displayed a different pattern of labelling including cells in the periventricular gray, the pontine reticular tegmental nucleus, the A-5 region, the medial vestibular complex, the prepositus hypoglossal nucleus, the parvicellular reticular nucleus, the lateral paragigantocellular nucleus, the raphe obscuras, the lateral reticular nucleus, and the lateral nucleus of the cerebellum. Immunostaining was seen in fibers within the red nucleus and within mossy fibers of the cerebellum. OMP levels could only be quantified by radioimmunoassay in the olfactory bulb of the three species and in the hamster cerebellum where they were 1/1,000 of those determined in the olfactory bulb. The authenticity of OMP measured in the RIA and detected immunocytochemically was verified by a double-antibody immunoisolation/immunodetection procedure, which confirmed that the antigen being visualized had the molecular properties expected for OMP. In summary, these experiments demonstrate that authentic OMP exists in small groups of neurons in many areas of the central nervous system.