We have examined the participation of a neuronal nitric oxide synthase (nNOS) signaling pathway in the elaboration of motor neuron dendrites during embryonic life. During chick embryogenesis, nNOS is expressed by interneurons that surround the motor neuron pools in the ventral horn. Pseudorabies virus tracing suggests that these cells, while juxtaposed to motor neurons are not synaptically connected to them. The downstream effectors, soluble guanylyl cyclase (sGC) and protein kinase G (PKG), are found in motor neurons as well as several other populations of spinal cord cells. To determine the functional significance of the nNOS/sGC/PKG signaling pathway, pharmacological inhibitors were applied to chick embryos and the effects on motor neuron dendrites monitored. Inhibition of nNOS activity led to a lasting reduction in the overall size and degree of branching of the dendritic tree. These alterations in dendritic architecture were also seen when the activity of sGC or PKG was blocked. Our results suggest that normal motor neuron dendrite elaboration depends, in part, on the activity-dependent generation of NO by ventral horn interneurons, which then activates sGC and PKG in motor neurons.