The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily. Two of the three major isoforms (NCAM 140 and NCAM 180) are transmembrane glycoproteins, which differ in their intracellular domains. The present study is concerned with the identification of novel intracellular binding partners of NCAM. We expressed and purified both cytoplasmic domains of NCAM. Using ligand affinity chromatography followed by peptide mass fingerprinting, we could identify several novel binding partners of the cytoplasmic domains of NCAM 140 and 180. We present data that alpha- and beta-tubulin as well as alpha-actinin I are associated with both NCAM 140 and 180. In contrast, beta-actin, tropomyosin, microtubuli-associated protein MAP 1A, and rhoA-binding kinase-alpha preferentially bind to NCAM 180. Furthermore, we demonstrate that inhibition of rhoA-binding kinase-alpha stimulates neurite outgrowth independently from NCAM. (C) 2003 Elsevier Inc. All rights reserved.