During development of the cerebellum, radial glial cells guide the migration of granule cell precursors from the external granular cell layer toward the internal granular cell layer. The cellular membranes of migrating neurons and glial fibers organize a specialized migration junction at the site of contact between these cells, and several molecules have been implicated in the control of this glial-guided neuronal migration program. The monoclonal antibody Jones (mAb Jones) recognizes the ganglioside 9-O-acetyl GD3, which is expressed in migratory profiles in the developing and adult CNS. Recently, this ganglioside was suggested to play a role in neuronal migration in cerebellar cultures. In this report, we use antibody perturbation assays to investigate a possible role of 9-O-acetyl GD3 in the neuronal migration program in vivo. The results show that chronic intracerebroventricular administration of mAb Jones arrests neuronal migration in the developing cerebellum of live animals. Proliferating granule cell precursors were labeled with 5-bromo-2'-deoxyuridine (BrdU), and their migratory behavior was analyzed and compared with control groups. Immunoblockage of 9-O-acetyl GD3 arrests 43% of the BrdU-labeled granule precursors in the external granular cell layer. Together with our previous results, this report strongly suggests that the ganglioside 9-O-acetyl GD3 plays a crucial role in the migration of cerebellar granule cells along radial glial fibers in the developing rat cerebellum.