The neural protein FE65 contains two types of protein-protein interaction modules: one WW binding domain and two phosphotyrosine binding domains. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the beta-amyloid precursor protein, which is implicated in Alzheimer disease. To understand the function of this adapter protein, we identified binding partners for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins of molecular masses 60, 75, 80, 140, and 200 kDa could be purified from mouse brain lysates by affinity to the FE65 WW domain. We identified two of these five proteins as the 80- and 140-kDa isoforms encoded by Mena, the mammalian homolog of the Drosophila Enabled gene. Using the SPOTs technique of peptide synthesis, we identified the sequences in Mena that interact with the FE65 WW domain and found that they contain the signature proline-proline-leucine-proline motif. Finally, we demonstrated that Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in beta-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular changes that underlie Alzheimer disease.