Cerebellar granule neurons in vitro specifically arrest the extension of their appropriate presynaptic axons, mossy fibers. This “stop- growing signal” may be an essential step in the formation and specificity of synapses. Here, we have tested whether ionotropic glutamate receptors are involved in the stop-growing signal. When explants of basilar pontine nuclei, a mossy fiber source, were cultured on granule neurons, most pontine neurites terminated <200 microm from their explant of origin, a criterion for the stop-growing signal. In contrast, treatment with the NMDA antagonist D(-)-2-amino-5- phosphonopentanoic acid (D-AP5) greatly increased the number of pontine neurites extending beyond 300 microm, whereas treatment with NMDA reduced the number of pontine neurites extending beyond 200 microm. A non-NMDA agonist (AMPA) and antagonist (6-cyano-7-nitroquinoxaline-2,3- dione) did not alter pontine neurite lengths. None of these agents affected neurite outgrowth from pontine explants in the absence of granule neurons, nor did any agent affect the survival of granule neurons. These results indicate that NMDA and D-AP5 specifically perturb an interaction between axons and target cells necessary for the stop-growing signal, and that NMDA receptors are critical for the development of a major cerebellar afferent system. These findings also suggest that NMDA-sensitive refinement of axon arbors during later development may involve the direct regulation of axon extension by target neurons.