We previously demonstrated that Fe65 protein is one of the ligands of the cytoplasmic domain of beta-amyloid precursor protein (APP). Another ligand of this molecule was recently identified; it is similar to Fe65, so it was named Fe65-like (Fe65L1). Herein we describe the cloning of another Fe65-like cDNA (Fe65L2), similar to Fe65 and to Fe65L1, which encodes a protein of approx. 50 kDa. Its cognate mRNA is expressed in various rat tissues, particularly in brain and testis. The three members of the Fe65 protein family share several structural and functional characteristics. The primary structures of the three proteins can be aligned in three regions corresponding to the protein-protein interaction domains of Fe65 [the protein-protein interaction domain containing two conserved tryptophan residues and the two phosphotyrosine interaction domain/phosphotyrosine binding (PID/PTB) domains], whereas the remaining sequences are poorly related. Like Fe65, Fe65L1 and Fe65L2 genes encode two different protein isoforms, derived from the alternative splicing of a very small exon of only six nucleotides, which results, within the N-terminal PID/PTB domain, in the presence or absence of two acidic/basic amino acids. Fe65L2 is able to interact, both in vitro and in vivo, with the intracellular domain of APP. Also, in the case of APP, another two closely related proteins exist, named beta-amyloid precursor-like protein (APLP)1 and APLP2: by using the interaction trap procedure we observed that both Fe65 and Fe65L2 interact with APP, APLP1 or APLP2, although with different efficiencies.