The occurrence, cellular distribution and nature of the 27-amino acid form of pituitary adenylate cyclase-activating peptide (PACAP-27) in the adrenal glands of mammals was investigated by means of immunohistochemistry, radioimmunoassay and chromatography. The concentrations (pmol/g wet weight) of PACAP-27-like immunoreactivity (LI) varied considerably between the species examined: mouse (n = 8), 12.1 ± 2.0; hamster (n = 4), 9.1 ± 1.5; rat (n = 13), 2.0 ± 0.3; cow (n = 3), 0.8 ± 0.1, and pig (n = 5), 0.7 ± 0.1. Upon HPLC, the immunoreactivity in extracts of rat, mouse and hamster adrenals coeluted with synthetic PACAP-27 while the immunoreactivity in extracts of cow and pig adrenals eluted as less hydrophobic material. Immunohistochemistry revealed the presence of PACAP-27-LI in the noradrenaline-storing chromaffin cells of the adrenal medulla. No nerve fibers exhibiting PACAP-27-LI could be detected. The effects of drug administration in vivo on the stores of PACAP-27-LI in the rat adrenal were studied and compared with the effects on the adrenal stores of neuropeptide Y (NPY)-LI. Splanchnic activation following insulin-induced hypoglycemia elicited a 41% depletion of NPY-LI 2 h after insulin injection, while the concentrations of PACAP-27-LI remained unchanged. Two days after reserpine administration the stores of PACAP-27-LI and NPY-LI were depleted by 62 and 41%, respectively. Two days later, the concentrations of NPY-LI were increased to 216% of controls, while the concentrations of PACAP-27-LI were similar to that of controls. Taken together with data from the literature, our findings suggest that PACAP may be involved in the regulation of adrenomedullary activity in more than one mammalian species. The stimuli that regulate the synthesis and release of PACAP-27 in the adrenal gland remains to be identified but seems to differ from those involved in the regulation of