Nitric oxide (NO) is a multifaceted messenger molecule believed to be involved in neural plasticity and development. Within the cerebellum, the NO synthesizing enzyme, NO synthase (NOS), is expressed exclusively by granule cells and stellate/basket neurons. In the adult cerebellum, levels of NOS expression can be used to define discrete clusters of granule cell populations. Differential expression of NOS by granule cells temporally coincides with the establishment of afferent innervation of granule cells. In primary cerebellar cultures that comprise a functional network of glutamatergic and GABAergic cerebellar neurons, blockade of electrical activity by tetrodotoxin induced the expression of the neuronal isoform of NOS (nNOS) in granule cells. Conversely, direct depolarization of cultured neurons with K+ completely downregulated nNOS expression. Suppression of NMDA receptor- and AMPA receptor-mediated spontaneous synaptic signaling in cultured cells resulted in a drastic upregulation of nNOS expression in granule neurons. In contrast, blockade of GABAA receptor-mediated intercellular communication did not affect nNOS expression by granule cells. Blocking N-, P-, and Q-type voltage-dependent Ca2+ channels resulted in a graded upregulation of NOS expression, whereas manipulations of the cAMP- dependent signal transduction pathway induced no changes. We conclude that nNOS expression in developing cerebellar granule cells is regulated by excitatory neurotransmission and that calcium is an important signal transduction molecule involved in this regulatory process.