Ablation of nonmuscle myosin heavy chain II-B (NMHC II-B) or expression of reduced amounts of R709C mutant NMHC II-B results in the development of hydrocephalus as early as embryonic day 13.5 (E13.5) in the mouse. Except for the abnormal protrusion of facial neurons into the developing fourth ventricle, no blockage of the cerebral ventricles is seen in these mice. In spite of this, all of the cerebral ventricles are enlarged in the mutant mice compared to wild-type mice. We, therefore, analyzed spinal cord development in various mutant mice including NMHC II-B knockout, R709C II-B mutant hypomorphic and non-hypomorphic mice, as well as mice expressing NMHC II-A in place of II-B. Disruption of the ventricular neuroepithelium was observed by E11.5 in both knockout and R709C mutant hypomorphic mice along the entire spinal cord, and neuroepithelial cells protruded into the developing central canal. Between E13.5-E14.5, the central canal was completely blocked. More severe abnormalities were seen in knockout mice compared to the R709C mutant hypomorphic mice. Interestingly, increased expression of mutant II-B to wild-type levels in non-hypomorphic mutant mice prevented blockage of the central canal and development of hydrocephalus, although these mice still suffered from novel abnormalities that were not seen in knockout and hypomorphic mutant mice. When NMHC II-A is expressed in place of II-B, no disruption of the neuroepithelium of the central canal or hydrocephalus was found in NMHC II-B ablated mice. These results indicate that NMHC II-A can replace NMHC II-B in maintaining the integrity of the neuroepithelium in the central canal, although other defects in the brain and heart were not rescued. In conclusion, NMHC II-B plays an important role in maintaining the integrity of the neuroepithelium in the spinal cord.