In order to construct a mouse brain-specific cDNA library, a novel subtraction approach exploiting two different restriction endonucleases was designed. In the strategy, an EcoRI/NotI adaptor was attached to cDNAs copied from liver and brain mRNAs. And EcoRI digested brain cDNA pool was subtracted with a large excess of NotI cleaved liver cDNAs. Then, the subtracted brain cDNA population was cloned into EcoRI site of vector DNA. A clone obtained by a random selection from the subtracted population was turned out to have a cDNA fragment expressed abundantly only in brain tissure as confirmed by Southern blot analysis. Based on the partial DNA sequence analysis of both ends of this insert fragment, it only contains a part of the corresponding cDNA sequence. In addition, the DNA sequence suggests the presence of two possible translational initiation sites contain sequences with 70＼% homology to Kozak sequence, the ribosome entry site in the eukaryotic system. Therefor, it is likely that the cloned cDNA fragment represents the 5``region of the cDNA. Homology search revealed no significant homology to reported sequences, suggesting that this fragment is a novel sequence unknown as yet.