Immunoreactive delta sleep-inducing peptide (IR-DSIP) has previously been localized to the ACTH/MSH cells of the human and porcine pituitary gland. In the present report, the distribution of IR-DSIP in the mouse pituitary gland was examined by immunocytochemistry. In this species, IR-DSIP was found to be co-localized with thyroid-stimulating hormone (TSH) in anterior pituitary thyrotrophs and was also present in nerve fibers in the posterior and intermediate lobes. The effect of synthetic DSIP on IR-ACTH release from dissociated mouse anterior pituitary cells was also studied. DSIP (>10<sup>–9</sup> M) inhibited both basal and CRF-induced IR-ACTH release from these cells. In addition, the effect of synthetic rat corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on IR-DSIP secretion was investigated. CRF and AVP at concentrations of 10<sup>–11–</sup>10<sup>–7</sup> M inhibited release of IR-DSIP from mouse anterior pituitary cells by 63%. When CRF and AVP (10<sup>–10</sup>–10<sup>–7</sup> M) were given concomitantly, the maximal inhibition of IR-DSIP release was observed at a concentration of 10<sup>–8</sup> M of CRF and AVP. However, these two peptides when given together showed no additive effect on IR-DSIP secretion. These findings suggest that during CRF induction of ACTH secretion from anterior pituitary corticotrophs, CRF may also act simultaneously to inhibit DSIP secretion from the thyrotrophs.