APCL, a homologue of the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin like APC. However, as its biological function remains unclear, we have been using a yeast two-hybrid system to search for proteins that associate with its carboxyl region. Among several cDNA clones we isolated from a fetal-brain cDNA library as candidates, six included an identical sequence with significant homology to EB1, a protein known to bind to APC. The full-length cDNA of this novel homologue of EB1, named EB3, encoded a protein of 282 amino acids with 54% identity to EB1, and it was expressed preferentially in brain tissue on Northern blots. Confocal microscopy demonstrated that exogenous EB3, like EB1, is associated with the cytoplasmic microtubule network. Moreover, in these experiments EB3 and APCL appeared together in the perinucleus and the cytoplasmic microtubule network. Since APCL is also expressed highly and specifically in the central nervous system, APCL-EB3 interaction may be specific to the CNS, possibly involving stability and/or extension of microtubules during neuritogenesis.