Expression of the neuropeptide galanin in the adrenal gland is rapidly induced by reflex stimulation of the splanchnic nerve following insulin-induced hypoglycemia. Here, we examine the cellular localization and mechanism of galanin mRNA upregulation in the adrenal gland after insulin administration, by Northern blot and in situ histochemical hybridization analysis. A 5- to 16-fold increase in galanin mRNA levels, measured by Northern blot hybridization using a rat galanin cRNA probe, was observed after insulin-induced hypoglycemia. An increase in galanin mRNA levels could be detected as early as two hours after administration of a single dose (10 U/kg) of insulin (Iletin II), consistent with the increase in galanin peptide levels in the adrenal gland within 24 h of insulin shock. Insulin-induced galanin mRNA upregulation was confined to the rat adrenal: neither hypothalamic nor pituitary levels of GAL mRNA were altered by insulin treatment. Adrenal galanin mRNA levels were maximally increased by 4 h, remained maximally elevated for at least 48 h, and had returned to baseline 6 days after insulin administration. In situ hybridization analysis localized galanin mRNA induction to scattered groups of chromaffin cells throughout the medulla. These data demonstrate that regulation of GAL biosynthesis in the adrenal medulla occurs at a pretranslational level, and in a subpopulation of chromaffin cells. Rapid and robust upregulation of galanin biosynthesis in chromaffin cells upon insulin-induced splanchnic nerve stimulation suggests a hormonal or paracrine role for galanin in the adrenomedullary response to hypoglycemic shock.