Netrins are a family of secreted proteins that function as tropic cues directing axon growth and cell migration during neural development. Beta-netrin or netrin-4 (NTN4), the fourth member of this family, was previously reported by Koch (M. Koch, J.R. Murrell, D.D. Hunter, P.F. Olson, W. Jin, D.R. Keene, W.J. Brunken, R.E. Burgeson. A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization. J. Cell Biol. 151 (2000) 221–234) and Yin (Y. Yin, J.R. Sanes, J.H. Miner, Identification and expression of mouse netrin-4. Mech. Dev. 96 (2000) 115–119), respectively. However, whether there is another isoform of netrin-4 and the expression patterns have not been fully determined. Here we report the cloning of a novel isoform of human netrin-4 using rapid amplification of complementary DNA (cDNA) ends (RACE) technique and analysis on the netrin-4 mRNA expression in adult rat brain using in situ hybridization. The new isoform lacks the signal peptide region and might represent a truncated form at the Laminin-N terminal domain of human netrin-4. Examination of the expression pattern of netrin-4 mRNA in adult rat brain revealed that the netrin-4 mRNA was widely expressed in many regions of the brain. High levels of netrin-4 mRNA was found in the pyramidal cell layer of the cerebral cortex, prepiriform cortex, amygdaloid nuclei, pyramidal layer of hippocampus, Purkinje's cells, medial cerebellar nucleus and interposed cerebellar nucleus, medial nucleus of the trapezoid body and mitral cell layer of the olfactory bulb. Moderate signals were present in frontal cortex, several thalamic and hypothalamic nuclei. The widespread expression of netrin-4 in adult rat brain indicates that netrin-4 is not only important for axon guidance during embryonic development, but also critical to neuronal plasticity in adult stage.