CNS neuronal migration is a specialized form of cell motility that sets forth the laminar structure of cortical regions of brain. To define the neuronal receptor systems in glial-guided neuronal migration, an in vitro assay was developed for mouse cerebellar granule neurons, which provides simultaneous tracking of hundreds of migrating neurons. Three general classes of receptor systems were analyzed, the neuron-glial adhesion ligand astrotactin, the neural cell adhesion molecules of the IgG superfamily, N-CAM, L1 and TAG-1, and the beta 1 subunit of the integrin family. In the absence of immune activities, migrating cerebellar granule neurons had an average in vitro migration rate of 12 microns h-1, with individual neurons exhibiting migration rates over a range between 0 to 70 microns h-1. The addition of anti-astrotactin antibodies (or Fabs) significantly reduced the mean rate of neuronal migration by sixty-one percent, resulting in eighty percent of the neurons having migration rates below 8 microns h-1. By contrast, blocking antibodies (or Fabs) against L1, N-CAM, TAG-1 or beta 1 integrin, individually or in combination, did not reduce the rate of neuronal migration. By video-enhanced contrast differential interference contrast microscopy the effects of anti-astrotactin antibodies were seen to be rapid. Within fifteen minutes of antibody application, streaming of cytoplasmic organelles into the leading process arrested, the nucleus shifted from a caudal to a central position, and the extension of filopodia and lamellopodia along the leading process ceased. Correlated video and electron microscopy suggested that the mechanism of arrest by antiastrotactin antibodies involved the failure to form new adhesion sites along the leading process and the disorganization of cytoskeletal components. These results suggest astrotactin acts as a neuronal receptor for granule neuron migration along astroglial fibers.