Any mutations in the human neural cell adhesion molecule L1 (hL1CAM) gene might cause various types of serious neurological syndromes in humans, characterized by increased mortality, mental retardation, and various malformations of the nervous system. Such missense mutations often cause severe abnormalities or even fatalities, and the reason for this may be a disruption of the adhesive function of L1CAM resulting from a misdirection of the degradative pathway. Transfection studies using neuroblastoma N2a cells demonstrated that hL1CAM carrying the missense mutations in the fibronectin-like type III (FnIII) domains most likely is located within the endoplasmic reticulum (ER), but it is less well expressed on the cell surface. One mutant, L935P, in the fourth FnIII domain, was chosen from six mutants (K655 and G698 at Fn1, L935P and P941 at Fn4, W1036 and Y1070 at Fn5) in the FnIII domains to study in detail the functions of hL1CAM200kDa, such as the intracellular traffic and degradation, because only a single band at 200 kDa was detected in the hL1CAML935P-transfected cells. hL1CAM200kDa is expressed predominantly in the ER but not on the cell surface. In addition, this missense mutated hL1CAM200kDa is polyubiquitylated at some sites in the extracellular domain and thus becomes degraded by proteasomes via the ER-associated degradation pathway. These observations demonstrate that the missense mutations of hL1CAM in the FnIII domain may cause the resultant pathogenesis because of a loss of expression on the cell surface resulting from misrouting to the degradative pathway. © 2011 Wiley-Liss, Inc.