Fyn tyrosine kinase is expressed extensively in the central nervous system (CNS) of mammals, and its genetic disruption in mouse displays several behavioral abnormalities with morphological and electrophysiological defects in the brain. To understand the signaling pathways in which Fyn is involved in the CNS, we screened molecules that directly associate with Fyn in neonatal mouse brain by using a two-hybrid yeast system. We isolated five cDNA clones with strong and reproducible Fyn-binding activity. Sequence analyses revealed that three of them are previously reported molecules, SON, tctex-1, and hnRNP K, and that two clones encode novel sequences. The hnRNP K has been shown to associate with Fyn, so our yeast system is appropriate to isolate Fyn-binding molecules. Northern hybridization analyses indicated that all isolated clones are expressed in the mouse brain and that the mRNA levels of the two molecules (tctex-1 and clone 82) change during development in the brain. A full-length cDNA of clone 82 was obtained and its deduced amino acid sequence was homologous to the RNA-binding proteins. Isolation of many Fyn-binding molecules suggest that, in the mouse CNS, Fyn mediates multiple signaling pathways by binding to multiple molecules and that some of these pathways play critical roles in determining a certain type of behavior.