The neural cell adhesion molecule (NCAM) is involved in neural development and in plasticity in the adult brain. NCAM140 and NCAM180 isoforms are transmembrane proteins with cytoplasmic domains that differ only in an alternatively spliced exon in the NCAM180 isoform. Both isoforms can interact with several extracellular and cytoplasmic molecules mediating NCAM-dependent functions. Most identified intracellular interaction partners bind to both isoforms, NCAM140 and NCAM180. To identify further intracellular interaction partners specifically binding to NCAM180 the cytosolic domain of human NCAM180 was recombinantly expressed and applied onto a protein macroarray containing the protein library from human fetal brain. We identified the ubiquitin C-terminal hydrolase (UCHL1) which has been described as a de-ubiquitinating enzyme as a potential interaction partner of NCAM180. Since NCAM180 and NCAM140 are ubiquitinated, NCAM140 was included in the subsequent experiments. A partial colocalization of both NCAM isoforms and UCHL1 was observed in primary neurons and the B35 neuroblastoma cell line. Overexpression of UCHL1 significantly decreased constitutive ubiquitination of NCAM180 and NCAM140 whereas inhibition of endogenous UCHL1 increased NCAM's ubiquitination. Furthermore, lysosomal localization of NCAM180 and NCAM140 was significantly reduced after overexpression of UCHL1 consistent with a partial colocalization of internalized NCAM with UCHL1. These data indicate that UCHL1 is a novel interaction partner of both NCAM isoforms that regulates their ubiquitination and intracellular trafficking.