Immunoreactive (IR) delta sleep-inducing peptide (DSIP) was examined by immunocytochemistry in the rat pituitary and adrenal gland and found to be colocalized with IR thyroid-stimulating hormone in the pituitary and with noradrenaline in the adrenal medulla. IR-DSIP was also detectable in nerve fibers in the posterior pituitary. By radioimmunoassay, IR-DSIP was quantified in plasma and tissue extracts after uni- or bilateral adrenalectomy. Significantly elevated plasma levels of IR-DSIP were measured 5 days after bilateral adrenalectomy (p < 0.001). IR-DSIP was increased (p < 0.05) in pituitary extracts from bilaterally adrenalectomized rats after 5 days, but not after 14 or 28 days. Sham- and unoperated animals did not significantly differ in plasma or tissue concentration of IR-DSIP. High-performance liquid chromatography of C<sub>18</sub> SEP-PAK® purified hypothalamus extracts revealed a single peak of IR-DSIP material of lower hydrophobicity than synthetic DSIP. The elevated concentration of IR-DSIP in the rat pituitary and plasma after bilateral adrenalectomy is consistent with the previously suggested role of DSIP to influence the activity of the hypothalamic-pituitary-adrenal axis.