A series of experiments was performed to monitor plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses to human gonadotropin-releasing hormone (GnRH) associated peptide (GAP) and related peptides. Ovariectomized hypothalamo-pituitary disconnected (HPD)ewes were challenged with injections (1–10 µg i.v.) of GAP, or given, with and without estradiol, hourly 500- or 1,000-ng pulses of GAP for 5–7 days. In all cases GAP failed to cause the release of LH or FSH from the pituitary gland or to alter mean plasma prolactin concentrations. When the same HPD ewes were given hourly or 2-hourly pulses of 250 ng GnRH, LH responded in a pulsatile manner, and FSH secretion was maintained, thus confirming the functional integrity of the pituitary gland after HPD. Fragments of the GAP molecule (pro-GnRH 14–36, 28–36, 38–49, and 51–66) and GAP dimer did not stimulate LH or FSH or inhibit prolactin release in HPD ewes. GAP and GAP dimer did not affect pituitary responsiveness to GnRH administration. GAP also failed to inhibit the thyrotropin-releasing hormone-induced rise in prolactin. Finally, GAP injections (100 µg i.v.) given to lactating ewes did not cause any change in plasma prolactin concentrations. These data show that human GAP, GAP dimer, or putative processed GAP peptides do not act on the sheep pituitary gland in a variety of physiological states to regulate gonadotropin or prolac