USING indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.