Vertebrate central nervous system (CNS) histogenesis depends on glia-guided migration of postmitotic neurons to form neuronal laminae. Previous studies have established that the neuronal protein astrotactin functions in murine cerebellar granule cell migration in vitro. The gene encoding astrotactin predicts a protein with three epidermal growth factor repeats and two fibronectin type III repeats. Astrotactin messenger RNA is expressed in postmitotic neuronal precursors in the cerebellum, hippocampus, cerebrum, and olfactory bulb, where migration establishes laminar structures. Fab fragments of antibodies to a recombinant astrotactin peptide blocked migration of cerebellar granule neurons in vitro along astroglial fibers. Transfection of astrotactin complementary DNA into 3T3 cells indicated that astrotactin acts as a ligand for neuron-glia binding during neuronal migration.