The complex morphological, electrophysiological and molecular properties of the adult vertebrate nervous system emerge over an extended period in prenatal and early postnatal life. Numerous studies have shown that synaptic activity plays a key role in the postnatal acquisition of mature neuronal phenotype. The cellular and molecular mechanisms subserving activity-dependent development are largely unknown. Several lines of evidence suggest that a rise in intracellular Ca2+ as a consequence of synaptic activity may regulate neuronal differentiation through its interactions with calcium-activated signal transduction molecules such as calcium/calmodulin kinase type II, protein kinase C or nitric oxide synthase (NOS). The aim of the present study is to identify potential signal transduction events subserving postnatal motor neuron development. Here we show that NOS antagonists block the molecular maturation of motor neurons and this effect is likely to be mediated by a subpopulation of ventral horn cells that express NOS transiently during early postnatal life. These results suggest that the local production of nitric oxide within the ventral horn may contribute to a late phase in motor neuron differentiation.