Nitric oxide synthase (NOS) expression is increased in peripheral sensory and central motor neurons after axotomy. By applying double-labelling immunofluorescence and non-radioactive in situ hybridization, we have investigated the regulation of NOS in axotomized sympathetic rat superior cervical ganglia. Furthermore, co-localization of NOS with vasoactive intestinal peptide, which is also induced by axotomy, has been examined. Very few (<0.1%) NOS-expressing neurons are observed in control ganglia. Some large cell bodies located at the exit of the internal carotid nerve are additionally immunoreactive for vasoactive intestinal peptide. One week following postganglionic axotomy, the number of NOS-immunoreactive and NOS mRNA-expressing neurons increases but does not exceed 2% of the whole neuronal population. About 20% of these neurons are also immunoreactive for vasoactive intestinal peptide. Preganglionic nerve fibre meshworks that are immunoreactive for NOS in untreated ganglia disappear after ganglionic decentralization, whereas some presumably postganglionic fibres remain visible after combined axotomy and decentralization. The findings are indicative of an increased synthesis of NOS in a small subset of postganglionic neurons of the rat superior cervical ganglion, possibly because of the loss of target-derived factors that inhibit nitric oxide synthesis under normal conditions.