TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.