NADPH-diaphorase reactivity and neuronal nitric oxide synthase (nNOS) immunostaining have been localised in sections of bovine adrenal glands. Both were present in nerve fibres and terminals in the subcapsular region and running between zona glomerulosa cells, amongst the medullary chromaffin cells, between large ganglion cells in rare encapsulated medullary ganglia and in large nerve bundles running through the cortex. Occasional isolated fibres were stained in deeper cortical layers. Both NADPH-diaphorase reactivity and nNOS immunoreactivity were present in a population of ganglion cells located individually or in small groups at the medullary-cortical boundary. NADPH-diaphorase reactivity was also found in all cortical cells (zona glomerulosa cells being more densely stained than other cortical cells) and in large fibrous structures in large nerve bundles (tentatively identified as glial cells): these structures were not stained with antisera to nNOS. Chromaffin cells were not stained with either technique. The possible role of neurally-released nitric oxide in the regulation of nerve-induced catecholamine secretion from chromaffin cells was investigated in isolated, perfused, bovine adrenal glands. The secretion of both adrenaline and noradrenaline in response to field stimulation of adrenal nerves at either 2 Hz or 10 Hz was unaffected by the presence of N-omega-nitro-L-arginine (30 mu M), sodium nitroprusside (10 mu M) or L-arginine (100 mu M) in the perfusing solution. It is concluded that, although nitric oxide may be generated and released from adrenal medullary nerves innervating chromaffin cells, it does not play a direct role in the acute regulation of adrenal catecholamine secretion.