We have investigated the possible presence of nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) in brainstem preganglionic parasympathetic neurons projecting to the sphenopalatine ganglion in rabbits, using combined retrograde axonal tracing and immunohistochemistry. Retrogradely labeled neurons were observed in the ipsilateral rostral medulla and caudal pons, in a region laterodorsal to the facial motor nucleus. Double-labeling experiments demonstrated that 75 +/- 5% of retrogradely labeled neurons contained NOS immunoreactivity, while all of retrogradely labeled neurons contained ChAT immunoreactivity. These observations suggest that nitric oxide could influence cholinergic transmission from preganglionic endings in the sphenopalatine ganglion.