A new mouse line has been produced in which the sixth Ig domain of the L1 cell adhesion molecule has been deleted. Despite the rather large deletion, L1 expression is preserved at normal levels. In vitro experiments showed that L1-L1 homophilic binding was lost, along with L1-alpha5beta1 integrin binding. However, L1-neurocan and L1-neuropilin binding were preserved and sema3a responses were intact. Surprisingly, many of the axon guidance defects present in the L1 knockout mice, such as abnormal corticospinal tract and corpus callosum, were not observed. Nonetheless, when backcrossed on the C57BL/6 strain, a severe hydrocephalus was observed and after several generations, became an embryonic lethal. These results imply that L1 binding to L1, TAG-1, or F3, and L1-alpha5beta1 integrin binding are not essential for normal development of a variety of axon pathways, and suggest that L1-L1 homophilic binding is important in the production of X-linked hydrocephalus.