Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.