We have isolated and characterized a novel cDNA encoding a small neuronal membrane protein showing high sequence homology to Neuro-p24/Neurensin-1, a protein containing a microtubule-associated domain at the carboxyl-terminus and exclusively localized to small vesicles of neurons. The newly identified Neurensin-2 constitutes two-membrane spanning domains but not the microtubule-binding domain, with a molecular mass of 28 kDa. Neurensin-2 mRNA is expressed only in brain, whereas the protein expressed in various neurons including those of the thalamus/hypothalamus and hippocampus, of postnatally developing mice. While the levels of Neurensin-1 mRNA and protein in retinoic acid-exposed mouse neuroblastoma Neuro2a cells increased, those of Neurensin-2 mRNA and protein remained unchanged. When the Neurensin-2 cDNA was transfected into Neuro2a cells, Neurensin-2 was expressed in small vesicles including lysosomes in the perinuclear region. On the cotransfection of Neurensin-1 and -2 cDNA into Neuro2a cells, Neurensin-2 was mainly found in small vesicles of the cell body and Neurensin-1 in those of growth cones. In nerve growth factor-stimulated PC12 cells, the intracellular localization of these proteins also differed. Furthermore, immunochemical staining of mouse brain revealed that Neurensin-1 and -2 had a similar distribution in many regions such as the Diagonal band, hippocampus, amygdaloid nucleus, and habenula nucleus, but differed in the intracellular localization as follows: Neurensin-1 was found mainly in neuritic processes, while Neurensin-2 was found in cell bodies. Thus, both Neurensin-1, and -2 are localized in small vesicles in neural cells, but their localizations of the vesicles are not always the same by each other, suggesting that they are under separate regulation.