Sympathetic (stellate and superior cervical ganglion) and sensory vagal (nodose and jugular ganglion) neurons innervating the guinea-pig trachea were labelled using a retrograde neuronal tracer (Fast Blue) and tested for immunoreactivity to nitric oxide synthase (NOS) and either tyrosine hydroxylase (TH; sympathetic ganglia) or substance P (SP; vagal afferent neurons). Approx. 3% of the sympathetic neurons innervating the trachea were NOS-positive. These neurons belonged to the non-catecholaminergic phenotype. Amongst the retrogradely labelled neurons in the vagal sensory ganglia, 5-10% of retrogradely labelled neurons in the nodose (inferior vagal) ganglion, and 10-20% of those in the jugular (superior vagal) ganglion were NOS-immunoreactive. All NOS-positive vagal afferent neurons labelled with retrograde tracer were negative for substance P. Accordingly, the results of these studies provide evidence that portions of the sympathetic and sensory innervation of the guinea-pig trachea is provided by NOS-immunoreactive neurons.